Human mitogenic oxidase

ABSTRACT

The present invention relates to new genes encoding for the production of novel proteins involved in generation of reactive oxygen intermediates that affect cell division. The present invention also provides vectors containing these genes, cells transfected with these vectors, antibodies raised against these novel proteins, kits for detection, localization and measurement of these genes and proteins, and methods to determine the activity of drugs to affect the activity of the proteins of the present invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 60/107,911, filed Nov. 10, 1998, U.S. Provisional PatentApplication Ser. No. 60/149,332, filed Aug. 17, 1999, and U.S.Provisional Patent Application Ser. No. 60/151,242, filed Aug. 27, 1999.Each of the aforementioned patent applications is hereby incorporated byreference in its entirety.

The U.S. Government has a paid-up license in this invention and theright in limited circumstances to require the patent owner to licenseothers on reasonable terms as provided for by the terms of NationalInstitutes of Health grants HL38206 and HL58000.

TECHNICAL FIELD

The present invention relates to the field of normal and abnormal cellgrowth, in particular mitogenic regulation. The present inventionprovides the following: nucleotide sequences encoding for the productionof enzymes that are mitogenic regulators; amino acid sequences of theseenzymes; vectors containing these nucleotide sequences; methods fortransfecting cells with vectors that produce these enzymes; transfectedcells; methods for administering these transfected cells to animals toinduce tumor formation; and antibodies to these enzymes that are usefulfor detecting and measuring levels of these enzymes, and for binding tocells possessing extracellular epitopes of these enzymes.

BACKGROUND OF THE INVENTION

Reactive oxygen intermediates (ROI) are partial reduction products ofoxygen: 1 electron reduces O₂ to form superoxide (O₂ ⁻), and 2 electronsreduce O₂ to form hydrogen peroxide (H₂O₂). ROI are generated as abyproduct of aerobic metabolism and by toxicological mechanisms. Thereis growing evidence for regulated enzymatic generation of O₂ ⁻ and itsconversion to H₂O₂ in a variety of cells. The conversion of O₂ ⁻ to H₂O₂occurs spontaneously, but is markedly accelerated by superoxidedismutase (SOD). High levels of ROI are associated with damage tobiomolecules such as DNA, biomembranes and proteins. Recent evidenceindicates generation of ROI under normal cellular conditions and pointsto signaling roles for O₂ ⁻ and H₂O₂.

Several biological systems generate reactive oxygen. Phagocytic cellssuch as neutrophils generate large quantities of ROI as part of theirbattery of bactericidal mechanisms. Exposure of neutrophils to bacteriaor to various soluble mediators such as formyl-Met-Leu-Phe or phorbolesters activates a massive consumption of oxygen, termed the respiratoryburst, to initially generate superoxide, with secondary generation ofH₂O₂, HOCl and hydroxyl radical. The enzyme responsible for this oxygenconsumption is the respiratory burst oxidase (nicotinamide adeninedinucleotide phosphate-reduced form (NADPH) oxidase).

There is growing evidence for the generation of ROI by non-phagocyticcells, particularly in situations related to cell proliferation.Significant generation of H₂O₂, O₂ ⁻ or both have been noted in somecell types. Fibroblasts and human endothelial cells show increasedrelease of superoxide in response to cytokines such as interleukin-1 ortumor necrosis factor (TNF) (Meier et al. (1989) Biochem J. 263,539-545.; Matsubara et al. (1986) J. Immun. 137, 3295-3298).Ras-transformed fibroblasts show increased superoxide release comparedwith control fibroblasts (Irani, et al. (1997) Science 275, 1649-1652).Rat vascular smooth muscle cells show increased H₂O₂ release in responseto PDGF (Sundaresan et al. (1995) Science 270, 296-299) and angiotensinII (Griendling et al. (1994) Circ. Res. 74, 1141-1148; Fukui et al.(1997) Circ. Res. 80, 45-51; Ushio-Fukai et al. (1996) J. Biol. Chem.271, 23317-23321), and H₂O₂ in these cells is associated with increasedproliferation rate. The occurrence of ROI in a variety of cell types issummarized in Table 1 (adapted from Burdon, R. (1995) Free Radical Biol.Med. 18, 775-794).

TABLE 1 Superoxide Hydrogen Peroxide human fibroblasts BaIb/3T3 cellshuman endothelial cells rat pancreatic islet cells human/rat smoothmuscle cells murine keratinocytes human fat cells rabbit chondrocyteshuman osteocytes human tumor cells BHK-21 cells fat cells, 3T3 L1 cellshuman colonic epithelial cells

ROI generated by the neutrophil have a cytotoxic function. While ROI arenormally directed at the invading microbe, ROI can also induce tissuedamage (e.g., in inflammatory conditions such as arthritis, shock, lungdisease, and inflammatory bowel disease) or may be involved in tumorinitiation or promotion, due to damaging effects on DNA. Nathan(Szatrowski et al. (1991) Canc. Res. 51, 794-798) proposed that thegeneration of ROI in tumor cells may contribute to the hypermutabilityseen in tumors, and may therefore contribute to tumor heterogeneity,invasion and metastasis.

In addition to cytotoxic and mutagenic roles, ROI have ideal propertiesas signal molecules: 1) they are generated in a controlled manner inresponse to upstream signals; 2) the signal can be terminated by rapidmetabolism of O₂ ⁻ and H₂O₂ by SOD and catalase/peroxidases; 3) theyelicit downstream effects on target molecules, e.g., redox-sensitiveregulatory proteins such as NF kappa B and AP-1 (Schreck et al. (1991)EMBO J. 10, 2247-2258; Schmidt et al. (1995) Chemistry & Biology 2,13-22). Oxidants such as O₂ ⁻ and H₂O₂ have a relatively well definedsignaling role in bacteria, operating via the SoxI/II regulon toregulate transcription.

ROI appear to have a direct role in regulating cell division, and mayfunction as mitogenic signals in pathological conditions related togrowth. These conditions include cancer and cardiovascular disease. O₂ ⁻is generated in endothelial cells in response to cytokines, and mightplay a role in angiogenesis (Matsubara et al. (1986) J. Immun. 137,3295-3298). O₂ ⁻ and H₂O₂ are also proposed to function as“life-signals”, preventing cells from undergoing apoptosis (Matsubara etal. (1986) J. Immun. 137, 3295-3298). As discussed above, many cellsrespond to growth factors (e.g., platelet derived growth factor (PDGF),epidermal derived growth factor (EGF), angiotensin II, and variouscytokines) with both increased production of O₂ ⁻/H₂O₂ and increasedproliferation. Inhibition of ROI generation prevents the mitogenicresponse. Exposure to exogenously generated O₂ ⁻ and H₂O₂ results in anincrease in cell proliferation. A partial list of responsive cell typesis shown below in Table 2 (adapted from Burdon, R. (1995) Free RadicalBiol. Med. 18, 775-794).

TABLE 2 Superoxide Hydrogen peroxide human, hamster fibroblasts mouseosteoblastic cells Balb/3T3 cells Balb/3T3 cells human histiocyticleukemia rat, hamster fibroblasts mouse epidermal cells human smoothmuscle cells rat colonic epithelial cells rat vascular smooth musclecells rat vascular smooth muscle cells

While non-transformed cells can respond to growth factors and cytokineswith the production of ROI, tumor cells appear to produce ROI in anuncontrolled manner. A series of human tumor cells produced largeamounts of hydrogen peroxide compared with non-tumor cells (Szatrowskiet al. (1991) Canc. Res. 51, 794-798). Ras-transformed NIH 3T3 cellsgenerated elevated amounts of superoxide, and inhibition of superoxidegeneration by several mechanisms resulted in a reversion to a “normal”growth phenotype.

O₂ ⁻ has been implicated in maintenance of the transformed phenotype incancer cells including melanoma, breast carcinoma, fibrosarcoma, andvirally transformed tumor cells. Decreased levels of the manganese formof SOD (MnSOD) have been measured in cancer cells and invitro-transformed cell lines, predicting increased O₂ ⁻ levels (Burdon,R. (1995) Free Radical Biol. Med. 18, 775-794). MnSOD is encoded onchromosome 6q25 which is very often lost in melanoma. Overexpression ofMnSOD in melanoma and other cancer cells (Church et al. (1993) Proc. ofNatl. Acad. Sci. 90, 3113-3117; Femandez-Pol et al. (1982) Canc. Res.42, 609-617; Yan et al. (1996) Canc. Res. 56, 2864-2871) resulted insuppression of the transformed phenotype.

ROI are implicated in growth of vascular smooth muscle associated withhypertension, atherosclerosis, and restenosis after angioplasty. O₂ ⁻generation is seen in rabbit aortic adventitia (Pagano et al. (1997)Proc. Natl. Acad. Sci. 94, 14483-14488). Vascular endothelial cellsrelease O₂ ⁻ in response to cytokines (Matsubara et al. (1986) J. Immun.137, 3295-3298). O₂ ⁻ is generated by aortic smooth muscle cells inculture, and increased O₂ ⁻ generation is stimulated by angiotensin IIwhich also induces cell hypertrophy. In a rat model system, infusion ofangiotensin II leads to hypertension as well as increased O₂ ⁻generation in subsequently isolated aortic tissue (Ushio-Fukai et al.(1996) J. Biol. Chem. 271, 23317-23321.; Yu et al. (1997) J. Biol. Chem.272, 27288-27294). Intravenous infusion of a form of SOD that localizesto the vasculature or an infusion of an O₂ ⁻ scavenger preventedangiotensin II induced hypertension and inhibited ROI generation (Fukuiet al. (1997) Circ. Res. 80, 45-51).

The neutrophil NADPH oxidase, also known as phagocyte respiratory burstoxidase, provides a paradigm for the study of the specialized enzymaticROI-generating system. This extensively studied enzyme oxidizes NADPHand reduces oxygen to form O₂ ⁻. NADPH oxidase consists of multipleproteins and is regulated by assembly of cytosolic and membranecomponents. The catalytic moiety consists of flavocytochrome b₅₅₈, anintegral plasma membrane enzyme comprised of two components: gp91phox(gp refers to glycoprotein; phox is an abbreviation of the wordsphagocyte and oxidase) and p22phox (p refers to protein). gp91phoxcontains 1 flavin adenine dinucleotide (FAD) and 2 hemes as well as theNADPH binding site. p22phox has a C-terminal proline-rich sequence whichserves as a binding site for cytosolic regulatory proteins. The twocytochrome subunits, gp91phox and p22phox appear to stabilize oneanother, since the genetic absence of either subunit, as in theinherited disorder chronic granulomatous disease (CGD), results in theabsence of the partner subunit (Yu et al. (1997) J. Biol. Chem. 272,27288-27294). Essential cytosolic proteins include p47phox, p67phox andthe small GTPase Rac, of which there are two isoforms. p47phox andp67phox both contain SH₃ regions and proline-rich regions whichparticipate in protein interactions governing assembly of the oxidasecomponents during activation. The neutrophil enzyme is regulated inresponse to bacterial phagocytosis or chemotactic signals byphosphorylation of p47phox, and perhaps other components, as well as byguanine nucleotide exchange to activate the GTP-binding protein Rac.

The origin of ROI in non-phagocytic tissues is unproven, but theoccurrence of phagocyte oxidase components has been evaluated in severalsystems by immunochemical methods, Northern blots and reversetranscriptase-polymerase chain reaction (RT-PCR). The message forp22phox is expressed widely, as is that for Rac1. Several cell typesthat are capable of O₂ ⁻ generation have been demonstrated to containall of the phox components including gp91phox, as summarized below inTable 3. These cell types include endothelial cells, aortic adventitiaand lymphocytes.

TABLE 3 Tissue gp91phox p22phox p47phox p67phox neutrophil +^(1,2)+^(1,2) +^(1,2) +^(1,2) aortic adventitia +¹   +¹   +¹   +¹  lymphocytes +²   +²   +^(1,2) +^(1,2) endothelial cells +²   +²  +^(1,2) +^(1,2) glomerular mesangial — +^(1,2) +^(1,2) +^(1,2) cellsfibroblasts — +²  +^(1,2) +²   aortic sm. muscle — +^(1,2) ? ? 1 =protein expression shown. 2 = mRNA expression shown.

However, a distinctly different pattern is seen in several other celltypes shown in Table 3 including glomerular mesangial cells, rat aorticsmooth muscle and fibroblasts. In these cells, expression of gp91phox isabsent while p22phox and in some cases cytosolic phox components havebeen demonstrated to be present. Since gp91phox and p22phox stabilizeone another in the neutrophil, there has been much speculation that somemolecule, possibly related to gp91phox, accounts for ROI generation inglomerular mesangial cells, rat aortic smooth muscle and fibroblasts(Ushio-Fukai et al. (1996) J. Biol. Chem. 271, 23317-23321).Investigation of fibroblasts from a patient with a genetic absence ofgp91phox provides proof that the gp91phox subunit is not involved in ROIgeneration in these cells (Emmendorffer et al. (1993) Eur. J. Haematol.51, 223-227). Depletion of p22phox from vascular smooth muscle using anantisense approach indicated that this subunit participates in ROIgeneration in these cells, despite the absence of detectable gp91phox(Ushio-Fukai et al. (1996) J. Biol. Chem. 271, 23317-23321). At thistime the molecular candidates possibly related to gp91phox and involvedin ROI generation in these cells are unknown.

Accordingly, what is needed is the identity of the proteins involved inROI generation, especially in non-phagocytic tissues and cells. What isalso needed are the nucleotide sequences encoding for these proteins,and the primary sequences of the proteins themselves. Also needed arevectors designed to include nucleotides encoding for these proteins.Probes and PCR primers derived from the nucleotide sequence are neededto detect, localize and measure nucleotide sequences, including mRNA,involved in the synthesis of these proteins. In addition, what is neededis a means to transfect cells with these vectors. What is also neededare expression systems for production of these molecules. Also neededare antibodies directed against these molecules for a variety of usesincluding localization, detection, measurement and passive immunization.

SUMMARY OF THE INVENTION

The present invention solves the problems described above by providing anovel family of nucleotide sequences and proteins encoded by thesenucleotide sequences termed mox proteins and duox proteins. Inparticular the present invention providess compositions comprising thenucleotide sequences SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:22, SEQ IDNO:41, SEQ ID NO:45, SEQ ID NO:47, and fragments thereof, which encodefor the expression of proteins comprising SEQ ID NO:2, SEQ ID NO:4, SEQID NO:21, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:48, respectively, andfragments thereof. While not wanting to be bound by the followingstatement, it is believed that these proteins are involved in ROIproduction. The present invention also provides vectors containing thesenucleotide sequences, cells transfected with these vectors which producethe proteins comprising SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:21, SEQ IDNO:42, SEQ ID NO:46, SEQ ID NO:48, and fragments thereof, and antibodiesto these proteins and fragments thereof. The present invention alsoprovides methods for stimulating cellular proliferation by administeringvectors encoded for production of the proteins comprising SEQ ID NO:2,SEQ ID NO:4, SEQ ID NO:21, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:48 andfragments thereof. The present invention also provides methods forstimulating cellular proliferation by administering the proteinscomprising SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:21, SEQ ID NO:42, SEQ IDNO:46, SEQ ID NO:48 and fragments thereof. The nucleotides andantibodies of the present invention are useful for the detection,localization and measurement of the nucleic acids encoding for theproduction of the proteins of the present invention, and also for thedetection, localization and measurement of the proteins of the presentinvention. These nucleotides and antibodies can be combined with otherreagents in kits for the purposes of detection, localization andmeasurement.

Most particularly, the present invention involves a method forregulation of cell division or cell proliferation by modifying theactivity or expression of the proteins described as SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:21, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:48 orfragments thereof. These proteins, in their naturally occurring orexpressed forms, are expected to be useful in drug development, forexample for screening of chemical and drug libraries by observinginhibition of the activity of these enzymes. Such chemicals and drugswould likely be useful as treatments for cancer, prostatic hypertrophy,benign prostatic hypertrophy, hypertension, atherosclerosis and manyother disorders involving abnormal cell growth or proliferation asdescribed below. The entire expressed protein may be useful in theseassays. Portions of the molecule which may be targets for inhibition ormodification include but are not limited to the binding site forpyridine nucleotides (NADPH or NADH), the flavoprotein domain(approximately the C-terminal 265 amino acids), and/or the binding orcatalytic site for flavin adenine dinucleotide (FAD).

The method of the present invention may be used for the development ofdrugs or other therapies for the treatment of conditions associated withabnormal growth including, but not limited to the following: cancer,psoriasis, prostatic hypertrophy, benign prostatic hypertrophy,cardiovascular disease, proliferation of vessels, including but notlimited to blood vessels and lymphatic vessels, arteriovenousmalformation, vascular problems associated with the eye,atherosclerosis, hypertension, and restenosis following angioplasty. Theenzymes of the present invention are excellent targets for thedevelopment of drugs and other agents which may modulate the activity ofthese enzymes. It is to be understood that modulation of activity mayresult in enhanced, diminished or absence of enzymatic activity.Modulation of the activity of these enzymes may be useful in treatmentof conditions associated with abnormal growth.

Drugs which affect the activity of the enzymes represented in SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:21, SEQ ID NO:42, SEQ ID NO:46, SEQ IDNO:48, or fragments thereof, may also be combined with othertherapeutics in the treatment of specific conditions. For example, thesedrugs may be combined with angiogenesis inhibitors in the treatment ofcancer, with antihypertensives for the treatment of hypertension, andwith cholesterol lowering drugs for the treatment of atherosclerosis.

Accordingly, an object of the present invention is to provide nucleotidesequences, or fragments thereof, encoding for the production ofproteins, or fragments thereof, that are involved in ROI production.

Another object of the present invention is to provide vectors containingthese nucleotide sequences, or fragments thereof.

Yet another object of the present invention is to provide cellstransfected with these vectors.

Still another object of the present invention is to administer cellstransfected with these vectors to animals and humans.

Another object of the present invention is to provide proteins, orfragments thereof, that are involved in ROI production.

Still another object of the present invention is to provide antibodies,including monoclonal and polyclonal antibodies, or fragments thereof,raised against proteins, or fragments thereof, that are involved in ROIproduction.

Another object of the present invention is to administer genescontaining nucleotide sequences, or fragments thereof, encoding for theproduction of proteins, or fragments thereof, that are involved in ROIproduction, to animals and humans and also to cells obtained fromanimals and humans.

Another object of the present invention is to administer antisensecomplimentary sequences of genes containing nucleotide sequences, orfragments thereof, encoding for the production of proteins, or fragmentsthereof, that are involved in ROI production, to animals and humans andalso to cells obtained from animals and humans.

Yet another object of the present invention is to provide a method forstimulating or inhibiting cellular proliferation by administeringvectors containing nucleotide sequences, or fragments thereof, encodingfor the production of proteins, or fragments thereof, that are involvedin ROI production, to animals and humans. It is also an object of thepresent invention to provide a method for stimulating or inhibitingcellular proliferation by administering vectors containing antisensecomplimentary sequences of nucleotide sequences, or fragments thereof,encoding for the production of proteins, or fragments thereof, that areinvolved in ROI production, to animals and humans. These methods ofstimulating cellular proliferation are useful for a variety of purposes,including but not limited to, developing animal models of tumorformation, stimulating cellular proliferation of blood marrow cellsfollowing chemotherapy or radiation, or in cases of anemia.

Still another object of the present invention is to provide antibodiesuseful in immunotherapy against cancers expressing the proteinsrepresented in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:21, SEQ ID NO:42, SEQID NO:46, SEQ ID NO:48 or fragments thereof.

Yet another object of the present invention is to provide nucleotideprobes useful for the detection, localization and measurement ofnucleotide sequences, or fragments thereof, encoding for the productionof proteins, or fragments thereof, that are involved in ROI production.

Another object of the present invention is to provide antibodies usefulfor the detection, localization and measurement of nucleotide sequences,or fragments thereof, encoding for the production of proteins, orfragments thereof, that are involved in ROI production.

Another object of the present invention is to provide kits useful fordetection of nucleic acids including the nucleic acids represented inSEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:22, SEQ ID NO:41, SEQ ID NO:45, SEQID NO:47, or fragments thereof, that encode for proteins, or fragmentsthereof, that are involved in ROI production.

Yet another object of the present invention is to provide kits usefulfor detection and measurement of nucleic acids including the nucleicacids represented in SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:22, SEQ IDNO:41, SEQ ID NO:45, SEQ ID NO:47, or fragments thereof, that encode forproteins, or fragments thereof, that are involved in ROI production.

Still another object of the present invention is to provide kits usefulfor the localization of nucleic acids including the nucleic acidsrepresented in SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:22, SEQ ID NO:41,SEQ ID NO:45, SEQ ID NO:47, or fragments thereof, that encode forproteins, or fragments thereof that are involved in ROI production.

Another object of the present invention is to provide kits useful fordetection of proteins, including the proteins represented in SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:21, SEQ ID NO:42, SEQ ID NO:46, SEQ IDNO:48, or fragments thereof, that are involved in ROI production.

Yet another object of the present invention is to provide kits usefulfor detection and measurement of proteins, including the proteinsrepresented in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:21, SEQ ID NO:42, SEQID NO:46, SEQ ID NO:48, or fragments thereof, that are involved in ROIproduction.

Still another object of the present invention is to provide kits usefulfor localization of proteins, including the proteins represented in SEQID NO:2, SEQ ID NO:4, SEQ ID NO:21, SEQ ID NO:42, SEQ ID NO:46, SEQ IDNO:48, or fragments thereof, that are involved in ROI production.

Yet another object of the present invention is to provides kits usefulfor the detection, measurement or localization of nucleic acids, orfragments thereof, encoding for proteins, or fragments thereof, that areinvolved in ROI production, for use in diagnosis and prognosis ofabnormal cellular proliferation related to ROI production.

Another object of the present invention is to provides kits useful forthe detection, measurement or localization of proteins, or fragmentsthereof, that are involved in ROI production, for use in diagnosis andprognosis of abnormal cellular proliferation related to ROI production.

These and other objects, features and advantages of the presentinvention will become apparent after a review of the following detaileddescription of the disclosed embodiments and the appended drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1(a-d). Comparison of amino acid sequences of the human mox1protein (labeled mox1.human, SEQ ID NO:2), rat mox1 protein (labeledmox1.rat, SEQ ID NO:21), human mox2 protein (labeled mox2.human., SEQ IDNO:4) of the present invention to human (gp 91phox/human.pep, SEQ IDNO:12) bovine (gp 91 phox/bovine.pep, SEQ ID NO:37), and murine (gp 91phox/mouse.pep, SEQ ID NO:38) proteins. Also included are related plantenzyme proteins cytb 558.arabidopsis.pep (SEQ ID NO:39) andcytb558.rice.pep, (SEQ ID NO:40). Enclosed in boxes are similar aminoacid residues.

FIG. 2. Sequence similarities among proteins related to gp91phoxincluding human mox1 (SEQ ID NO:2), human mox2 (SEQ ID NO:4), and ratmox1 (SEQ ID NO:21). The dendrogram indicates the degree of similarityamong this family of proteins, and also includes the related plantenzymes.

FIG. 3. Cell free assay for mox-1 activity. Superoxide generation wasmeasured using the chemiluminescent reaction between lucigenin andsuperoxide in cell lysates from vector control NEF2 and mox1 transfectedNIH3T3 cells.

FIG. 4. Superoxide generation by human mox1. Reduction of NBT in mox1transfected and control fibroblasts was measured in the absence (filledbars) or presence (open bars) or superoxide dismutase.

FIG. 5. Aconitase (filled bars), lactate dehydrogenase (narrow hatching)and fumarase (broad hatching) were determined in lysates of cellstransfected with vector alone (NEF2) or with mox1 (YA26, YA28 andYA212).

DETAILED DESCRIPTION OF THE INVENTION

The present invention solves the problems described above by providing anovel family of nucleotide sequences and proteins, encoded by thesenucleotide sequences, termed mox proteins and duox proteins. The term“mox” refers to “mitogenic oxidase” while the term “duox” refers to“dual oxidase”. In particular, the present invention provides novelcompositions comprising the nucleotide sequences SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:22, SEQ ID NO:41, SEQ ID NO:45, SEQ ID NO:47, andfragments thereof, which encode, respectively, for the expression ofproteins comprising SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:21, SEQ IDNO:42, SEQ ID NO:46, SEQ ID NO:48 and fragments thereof.

Both the mox and duox proteins described herein have homology to thegp91phox protein involved in ROI generation, however, the mox and duoxproteins comprise a novel and distinct family of proteins. The moxproteins included in the present invention have a molecular weight ofapproximately 65 kDa as determined by reducing gel electrophoresis andare capable of inducing ROI generation in cells. As described in moredetail below, the mox proteins of the present invention also function inthe regulation of cell growth, and are therefore implicated in diseasesinvolving abnormal cell growth such as cancer. The present inventiondescribes mox proteins found in human and rat, however, it is likelythat the mox family of genes/proteins is widely distributed amongmulticellular organisms.

The duox proteins described herein are larger than the mox proteins andhave three distinct regions: the amino terminal region having homologyto peroxidase proteins, the internal region having homology tocalmodulin (CAM) proteins and the carboxy-terminal region havinghomology to mox proteins. Human duox1 is shown in SEQ ID NO:46 and aportion of human duox2 is shown in SEQ ID NO:48. Nucleotides encodingduoxt and duox2 proteins are also shown in SEQ ID NO: 45 and SEQ IDNO:47, respectively. In addition to the human duox proteins, comparisonof the sequence of human duox1 and human duox2 with genomic databasesusing BLAST searching resulted in the identification of two homologs ofduox in C. elegans (Ce-duox1 and Ce-duox2). Drosophila also appears tohave at least one duox homolog. Thus, the duox family of genes/proteinsis widely distributed among multicellular organisms.

Although not wanting to be bound by the following statement, it isbelieved that duox1 and duox2 have dual enzymatic functions, catalyzingboth the generation of superoxide and peroxidative type reactions. Thelatter class of reactions utilize hydrogen peroxide as a substrate (andin some cases have been proposed to utilize superoxide as a substrate).Since hydrogen peroxide is generated spontaneously from the dismutationof superoxide, it is believed that the NAD(P)H oxidase domain generatesthe superoxide and/or hydrogen peroxide which can then be used as asubstrate for the peroxidase domain. In support of this hypothesis, amodel for the duox1 protein in C. elegans has been developed that has anextracellular N-terminal peroxidase domain, a transmembrane region and aNADPH binding site located on the cytosolic face of the plasma membrane.By analogy with the neutrophil NADPH-oxidase which generatesextracellular superoxide, human duox1 is predicted to generatesuperoxide and its byproduct hydrogen peroxide extracellularly where itcan be utilized by the peroxidase domain.

While the ROI generated by duox1 and duox2 may function as does mox1 inregulation of cell growth, the presence of the peroxidase domain islikely to confer additional biological functions. Depending upon theco-substrate, peroxidases can participate in a variety of reactionsincluding halogenation such as the generation of hypochlorous acid(HOCl) by myeloperoxidase and the iodination of tyrosine to formthyroxin by thyroid peroxidase. Peroxidases have also been documented toparticipate in the metabolism of polyunsaturated fatty acids, and in thechemical modification of tyrosine in collagen (by sea urchinovoperoxidase). Although not wanting to be bound by this statement, itis believed that the predicted transmembrane nature of duox1 facilitatesits function in the formation or modification of extracellular matrix orbasement membrane. Since the extracellular matrix plays an importantrole in tumor cell growth, invasion and metastasis, it is believed thatthe duox type enzymes play a pathogenic role in such conditions.

In addition to the nucleotide sequences described above, the presentinvention also provides vectors containing these nucleotide sequencesand fragments thereof, cells transfected with these vectors whichproduce the proteins comprising SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:21,SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:48 and fragments thereof, andantibodies to these proteins and fragments thereof. The presentinvention also provides methods for stimulating cellular proliferationby administering vectors, or cells containing vectors, encoded forproduction of the proteins comprising SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:21, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:48 and fragments thereof.The nucleotides and antibodies of the present invention are useful forthe detection, localization and measurement of the nucleic acidsencoding for the production of the proteins of the present invention,and also for the detection, localization and measurement of the proteinsof the present invention. These nucleotides and antibodies can becombined with other reagents in kits for the purposes of detection,localization and measurement. These kits are useful for diagnosis andprognosis of conditions involving cellular proliferation associated withproduction of reactive oxygen intermediates.

The present invention solves the problems described above by providing acomposition comprising the nucleotide sequence SEQ ID NO:1 and fragmentsthereof. The present invention also provides a composition comprisingthe nucleotide sequence SEQ ID NO:3 and fragments thereof. The presentinvention also provides a composition comprising the nucleotide sequenceSEQ ID NO:22 and fragments thereof. The present invention also providesa composition comprising the nucleotide sequence SEQ ID NO:41 andfragments thereof. The present invention also provides a compositioncomprising the nucleotide sequence SEQ ID NO:45 and fragments thereof.The present invention also provides a composition comprising thenucleotide sequence SEQ ID NO:47 and fragments thereof.

The present invention provides a composition comprising the protein SEQID NO:2 encoded by the nucleotide sequence SEQ ID NO:1. The presentinvention provides a composition comprising the protein SEQ ID NO:4encoded by the nucleotide sequence SEQ ID NO:3. The present inventionprovides a composition comprising the protein SEQ ID NO:21 encoded bythe nucleotide sequence SEQ ID NO:22. The present invention provides acomposition comprising the protein SEQ ID NO:42 encoded by thenucleotide sequence SEQ ID NO:41. The present invention provides acomposition comprising the protein SEQ ID NO:46 encoded by thenucleotide sequence SEQ ID NO:45. The present invention provides acomposition comprising the protein SEQ ID NO:48 encoded by thenucleotide sequence SEQ ID NO:47.

The present invention provides a composition comprising the protein SEQID NO:2 or fragments thereof, encoded by the nucleotide sequence SEQ IDNO:1 or fragments thereof. The present invention also provides acomposition comprising the protein SEQ ID NO:4 or fragments thereof,encoded by the nucleotide sequence SEQ ID NO:3 or fragments thereof. Thepresent invention also provides a composition comprising the protein SEQID NO:21 or fragments thereof, encoded by the nucleotide sequence SEQ IDNO:22 or fragments thereof. The present invention also provides acomposition comprising the protein SEQ ID NO:42 or fragments thereof,encoded by the nucleotide sequence SEQ ID NO:41 or fragments thereof.The present invention also provides a composition comprising the proteinSEQ ID NO:46 or fragments thereof, encoded by the nucleotide sequenceSEQ ID NO:45 or fragments thereof. The present invention also provides acomposition comprising the protein SEQ ID NO:48 or fragments thereof,encoded by the nucleotide sequence SEQ ID NO:47 or fragments thereof.

The present invention also provides vectors containing the nucleotidesequences SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:22, SEQ ID NO:41, SEQ IDNO:45, SEQ ID NO:47 or fragments thereof. The present invention alsoprovides cells transfected with these vectors. In addition, the presentinvention provides cells stably transfected with the nucleotide sequenceSEQ ID NO:1 or fragments thereof. The present invention also providescells stably transfected with the nucleotide sequence SEQ ID NO:3 orfragments thereof. The present invention also provides cells stablytransfected with the nucleotide sequence SEQ ID NO:22 or fragmentsthereof. The present invention also provides cells stably transfectedwith the nucleotide sequence SEQ ID NO:41 or fragments thereof. Thepresent invention also provides cells stably transfected with thenucleotide sequence SEQ ID NO:45 or fragments thereof. The presentinvention also provides cells stably transfected with the nucleotidesequence SEQ ID NO:47 or fragments thereof.

The present invention provides cells stably transfected with thenucleotide sequence SEQ ID NO:1 or fragments thereof, which produce theprotein SEQ ID NO:2 or fragments thereof. In addition, the presentinvention provides cells stably transfected with the nucleotide sequenceSEQ ID NO:3 or fragments thereof which produce the protein SEQ ID NO:4or fragments thereof. In addition, the present invention provides cellsstably transfected with the nucleotide sequence SEQ ID NO:22 orfragments thereof which produce the protein SEQ ID NO:21 or fragmentsthereof. The present invention also provides cells stably transfectedwith the nucleotide sequence SEQ ID NO:41 or fragments thereof whichproduce the protein SEQ ID NO:42 or fragments thereof. The presentinvention also provides cells stably transfected with the nucleotidesequence SEQ ID NO:45 or fragments thereof which produce the protein SEQID NO:46 or fragments thereof. The present invention also provides cellsstably transfected with the nucleotide sequence SEQ ID NO:47 orfragments thereof which produce the protein SEQ ID NO:48 or fragmentsthereof.

The present invention provides a method for stimulating growth byadministering cells stably transfected with the nucleotide sequence SEQID NO:1 which produce the protein SEQ ID NO:2 or fragments thereof. Thepresent invention also provides a method for stimulating growth byadministering cells stably transfected with the nucleotide sequence SEQID NO:3 or fragments thereof, which produce the protein SEQ ID NO:4 orfragments thereof. The present invention also provides a method forstimulating growth by administering cells stably transfected with thenucleotide sequence SEQ ID NO:22 or fragments thereof, which produce theprotein SEQ ID NO:21 or fragments thereof. The present invention alsoprovides a method for stimulating growth by administering cells stablytransfected with the nucleotide sequence SEQ ID NO:41 or fragmentsthereof, which produce the protein SEQ ID NO:42 or fragments thereof.The present invention also provides a method for stimulating growth byadministering cells stably transfected with the nucleotide sequence SEQID NO:45 or fragments thereof, which produce the protein SEQ ID NO:46 orfragments thereof. The present invention also provides a method forstimulating growth by administering cells stably transfected with thenucleotide sequence SEQ ID NO:47 or fragments thereof, which produce theprotein SEQ ID NO:48 or fragments thereof.

Specifically, the present invention provides a method for stimulatingtumor formation by administering cells stably transfected with thenucleotide sequence SEQ ID NO:1 or fragments thereof, which produce theprotein SEQ ID NO:2 or fragments thereof. The present invention alsoprovides a method for stimulating tumor formation by administering cellsstably transfected with the nucleotide sequence SEQ ID NO:3 or fragmentsthereof, which produce the protein SEQ ID NO:4 or fragments thereof. Thepresent invention also provides a method for stimulating tumor formationby administering cells stably transfected with the nucleotide sequenceSEQ ID NO:22 or fragments thereof, which produce the protein SEQ IDNO:21 or fragments thereof. The present invention also provides a methodfor stimulating tumor formation by administering cells stablytransfected with the nucleotide sequence SEQ ID NO:41 or fragmentsthereof, which produce the protein SEQ ID NO:42 or fragments thereof.The present invention also provides a method for stimulating tumorformation by administering cells stably transfected with the nucleotidesequence SEQ ID NO:45 or fragments thereof, which produce the proteinSEQ ID NO:46 or fragments thereof. The present invention also provides amethod for stimulating tumor formation by administering cells stablytransfected with the nucleotide sequence SEQ ID NO:47 or fragmentsthereof, which produce the protein SEQ ID NO:48 or fragments thereof.

The present invention may also be used to develop anti-sense nucleotidesequences to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:22, SEQ ID NO:41, SEQID NO:45, SEQ ID NO:47 or fragments thereof. These anti-sense moleculesmay be used to interfere with translation of nucleotide sequences, suchas SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:22, SEQ ID NO:41, SEQ ID NO:45,SEQ ID NO:47, or fragments thereof, that encode for proteins such as SEQID NO:2, SEQ ID NO:4, SEQ ID NO:21, SEQ ID NO:42, SEQ ID NO:46, SEQ IDNO:48 or fragments thereof. Administration of these anti-sensemolecules, or vectors encoding for anti sense molecules, to humans andanimals, would interfere with production of proteins such as SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:21, SEQ ID NO:42, SEQ ID NO:46, SEQ IDNO:48, or fragments thereof, thereby decreasing production of ROIs andinhibiting cellular proliferation. These methods are useful in producinganimal models for use in study of tumor development and vascular growth,and for study of the efficacy of treatments for affecting tumor andvascular growth in vivo.

The present invention also provides a method for high throughputscreening of drugs and chemicals which modulate the proliferativeactivity of the enzymes of the present invention, thereby affecting celldivision. Combinatorial chemical libraries may be screened for chemicalswhich modulate the proliferative activity of these enzymes. Drugs andchemicals may be evaluated based on their ability to modulate theenzymatic activity of the expressed or endogenous proteins, includingthose represented by SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:21, SEQ IDNO:42, SEQ ID NO:46, SEQ ID NO:48 or fragments thereof. Endogenousproteins may be obtained from many different tissues or cells, such ascolon cells. Drugs may also be evaluated based on their ability to bindto the expressed or endogenous proteins represented by SEQ ID NO:2, SEQID NO:4, SEQ ID NO:21, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:48 orfragments thereof. Enzymatic activity may be NADPH- or NADH-dependentsuperoxide generation catalyzed by the holoprotein. Enzymatic activitymay also be NADPH- or NADH-dependent diaphorase activity catalyzed byeither the holoprotein or the flavoprotein domain.

By flavoprotein domain, is meant approximately the C-terminal half ofthe enzymes shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:21, SEQ IDNO:42, or fragments thereof, and the C-terminal end of the enzymes shownin SEQ ID NO:46 and SEQ ID NO:48 (approximately the C-terminal 265 aminoacids). This fragment of gp91phox has NADPH-dependent reductase activitytowards cytochrome c, nitrobluetetrazolium and other dyes. Expressedproteins or fragments thereof can be used for robotic screens ofexisting combinatorial chemical libraries. While not wanting to be boundby the following statement, it is believed that the NADPH or NADHbinding site and the FAD binding site are useful for evaluating theability of drugs and other compositions to bind to the mox and duoxenzymes or to modulate their enzymatic activity. The use of theholoprotein or the C-terminal half or end regions are preferred fordeveloping a high throughput drug screen. Additionally, the N-terminalone-third of the duox domain (the peroxidase domain) may also be used toevaluate the ability of drugs and other compositions to inhibit theperoxidase activity, and for further development of a high throughputdrug screen.

The present invention also provides antibodies directed to the proteinsSEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:21, SEQ ID NO:42, SEQ ID NO:46, SEQID NO:48 and fragments thereof. The antibodies of the present inventionare useful for a variety of purposes including localization, detectionand measurement of the proteins SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:21,SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:48 and fragments thereof. Theantibodies may be employed in kits to accomplish these purposes. Theseantibodies may also be linked to cytotoxic agents for selected killingof cells. The term antibody is meant to include any class of antibodysuch as IgG, IgM and other classes. The term antibody also includes acompletely intact antibody and also fragments thereof, including but notlimited to Fab fragments and Fab+Fc fragments.

The present invention also provides the nucleotide sequences SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:22, SEQ ID NO:41, SEQ ID NO:45, SEQ IDNO:47 and fragments thereof. These nucleotides are useful for a varietyof purposes including localization, detection, and measurement ofmessenger RNA involved in synthesis of the proteins represented as SEQID NO:2, SEQ ID NO:4, SEQ ID NO:21, SEQ ID NO:42, SEQ ID NO:46, SEQ IDNO:48 and fragments thereof. These nucleotides may also be used in theconstruction of labeled probes for the localization, detection, andmeasurement of nucleic acids such as messenger RNA or alternatively forthe isolation of larger nucleotide sequences containing the nucleotidesequences shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:22, SEQ ID NO:41,SEQ ID NO:45, SEQ ID NO:47 or fragments thereof. These nucleotidesequences may be used to isolate homologous strands from other speciesusing techniques known to one of ordinary skill in the art. Thesenucleotide sequences may also be used to make probes and complementarystrands. In particular, the nucleotide sequence shown in SEQ ID NO:47may be used to isolate the complete coding sequence for duox2. Thenucleotides may be employed in kits to accomplish these purposes.

Most particularly, the present invention involves a method formodulation of growth by modifying the proteins represented as SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:21, SEQ ID NO:42, SEQ ID NO:46, SEQ IDNO:48 or fragments thereof.

The term “mitogenic regulators” is used herein to mean any molecule thatacts to affect cell division.

The term “animal” is used herein to mean humans and non-human animals ofboth sexes.

The terms “a”, “an” and “the” as used herein are defined to mean one ormore and include the plural unless the context is inappropriate.

“Proteins”, “peptides,” “polypeptides” and “oligopeptides” are chains ofamino acids (typically L-amino acids) whose alpha carbons are linkedthrough peptide bonds formed by a condensation reaction between thecarboxyl group of the alpha carbon of one amino acid and the amino groupof the alpha carbon of another amino acid. The terminal amino acid atone end of the chain (i.e., the amino terminal) has a free amino group,while the terminal amino acid at the other end of the chain (i. e., thecarboxy terminal) has a free carboxyl group. As such, the term “aminoterminus” (abbreviated N-terminus) refers to the free alpha-amino groupon the amino acid at the amino terminal of the protein, or to thealpha-amino group (imino group when participating in a peptide bond) ofan amino acid at any other location within the protein. Similarly, theterm “carboxy terminus” (abbreviated C-terminus) refers to the freecarboxyl group on the amino acid at the carboxy terminus of a protein,or to the carboxyl group of an amino acid at any other location withinthe protein.

Typically, the amino acids making up a protein are numbered in order,starting at the amino terminal and increasing in the direction towardthe carboxy terminal of the protein. Thus, when one amino acid is saidto “follow” another, that amino acid is positioned closer to the carboxyterminal of the protein than the preceding amino acid.

The term “residue” is used herein to refer to an amino acid (D or L) oran amino acid mimetic that is incorporated into a protein by an amidebond. As such, the amino acid may be a naturally occurring amino acidor, unless otherwise limited, may encompass known analogs of naturalamino acids that function in a manner similar to the naturally occurringamino acids (i.e., amino acid mimetics). Moreover, an amide bond mimeticincludes peptide backbone modifications well known to those skilled inthe art.

Furthermore, one of skill will recognize that, as mentioned above,individual substitutions, deletions or additions which alter, add ordelete a single amino acid or a small percentage of amino acids(typically less than 5%, more typically less than 1%) in an encodedsequence are conservatively modified variations where the alterationsresult in the substitution of an amino acid with a chemically similaramino acid. Conservative substitution tables providing functionallysimilar amino acids are well known in the art. The following six groupseach contain amino acids that are conservative substitutions for oneanother:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

When the peptides are relatively short in length (i.e., less than about50 amino acids), they are often synthesized using standard chemicalpeptide synthesis techniques. Solid phase synthesis in which theC-terminal amino acid of the sequence is attached to an insolublesupport followed by sequential addition of the remaining amino acids inthe sequence is a preferred method for the chemical synthesis of theantigenic epitopes described herein. Techniques for solid phasesynthesis are known to those skilled in the art.

Alternatively, the antigenic epitopes described herein are synthesizedusing recombinant nucleic acid methodology. Generally, this involvescreating a nucleic acid sequence that encodes the peptide or protein,placing the nucleic acid in an expression cassette under the control ofa particular promoter, expressing the peptide or protein in a host,isolating the expressed peptide or protein and, if required, renaturingthe peptide or protein. Techniques sufficient to guide one of skillthrough such procedures are found in the literature.

When several desired protein fragments or peptides are encoded in thenucleotide sequence incorporated into a vector, one of skill in the artwill appreciate that the protein fragments or peptides may be separatedby a spacer molecule such as, for example, a peptide, consisting of oneor more amino acids. Generally, the spacer will have no specificbiological activity other than to join the desired protein fragments orpeptides together, or to preserve some minimum distance or other spatialrelationship between them. However, the constituent amino acids of thespacer may be selected to influence some property of the molecule suchas the folding, net charge, or hydrophobicity. Nucleotide sequencesencoding for the production of residues which may be useful inpurification of the expressed recombinant protein may be built into thevector. Such sequences are known in the art. For example, a nucleotidesequence encoding for a poly histidine sequence may be added to a vectorto facilitate purification of the expressed recombinant protein on anickel column.

Once expressed, recombinant peptides, polypeptides and proteins can bepurified according to standard procedures known to one of ordinary skillin the art, including ammonium sulfate precipitation, affinity columns,column chromatography, gel electrophoresis and the like. Substantiallypure compositions of about 50 to 99% homogeneity are preferred, and 80to 95% or greater homogeneity are most preferred for use as therapeuticagents.

One of skill in the art will recognize that after chemical synthesis,biological expression or purification, the desired proteins, fragmentsthereof and peptides may possess a conformation substantially differentthan the native conformations of the proteins, fragments thereof andpeptides. In this case, it is often necessary to denature and reduceprotein and then to cause the protein to re-fold into the preferredconformation. Methods of reducing and denaturing proteins and inducingre-folding are well known to those of skill in the art.

The genetic constructs of the present invention include coding sequencesfor different proteins, fragments thereof, and peptides. The geneticconstructs also include epitopes or domains chosen to permitpurification or detection of the expressed protein. Such epitopes ordomains include DNA sequences encoding the glutathione binding domainfrom glutathione S-transferase, hexa-histidine, thioredoxin,hemagglutinin antigen, maltose binding protein, and others commonlyknown to one of skill in the art. The preferred genetic constructincludes the nucleotide sequences of SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:22, SEQ ID NO:41, SEQ ID NO:45, SEQ ID NO:47 or fragments thereof. Itis to be understood that additional or alternative nucleotide sequencesmay be included in the genetic constructs in order to encode for thefollowing: a) multiple copies of the desired proteins, fragmentsthereof, or peptides; b) various combinations of the desired proteins,fragments thereof, or peptides; and c) conservative modifications of thedesired proteins, fragments thereof, or peptides, and combinationsthereof. Preferred proteins include the human mox1 protein and humanmox2 protein shown as SEQ ID NO:2 and SEQ ID NO:4, respectively, andfragments thereof. Some preferred fragments of the human mox1 protein(SEQ ID NO:2) include but are not limited to the proteins shown as SEQID NO:23, SEQ ID NO:24, and SEQ ID NO:25. The protein mox1 is alsocalled p65mox in this application. Another preferred protein of thepresent invention is rat mox1 protein shown as SEQ ID NO:21 andfragments thereof. Another preferred protein of the present invention israt mox1B protein shown as SEQ ID NO:42 and fragments thereof. Yetanother preferred protein of the present invention is duox1 proteinshown as SEQ ID NO:46 and fragments thereof. Still another preferredprotein of the present invention is duox2 protein. A partial amino acidsequence of the duox2 protein is shown as SEQ ID NO:48.

The nucleotide sequences of the present invention may also be employedto hybridize to nucleic acids such as DNA or RNA nucleotide sequencesunder high stringency conditions which permit detection, for example, ofalternately spliced messages.

The genetic construct is expressed in an expression system such as inNIH 3T3 cells using recombinant, sequences in a pcDNA-3 vector(Invitrogen, Carlsbad, Calif.) to produce a recombinant protein.Preferred expression systems include but are not limited to Cos-7 cells,insect cells using recombinant baculovirus, and yeast. It is to beunderstood that other expression systems known to one of skill in theart may be used for expression of the genetic constructs of the presentinvention. The preferred proteins of the present invention are theproteins referred to herein as human mox1 and human mox2 or fragmentsthereof which have the amino acid sequences set forth in SEQ ID NO:3 andSEQ ID NO:4, respectively, or an amino acid sequence having amino acidsubstitutions as defined in the definitions that do not significantlyalter the function of the recombinant protein in an adverse manner.Another preferred protein of the present invention is referred to hereinas rat mox1 and has the amino acid sequence set forth in SEQ ID NO:21.Yet another preferred protein of the present invention is referred toherein as rat mox1 B and has the amino acid sequence set forth in SEQ IDNO:42. Two other preferred proteins of the present invention arereferred to herein as human duox1 and human duox2, or fragments thereof,which have the amino acid sequences set forth in SEQ ID NO:46 and SEQ IDNO:48, respectively, or an amino acid sequence having amino acidsubstitutions as defined in the definitions that do not significantlyalter the function of the recombinant protein in an adverse manner.

Terminology

It should be understood that some of the terminology used to describethe novel mox and duox proteins contained herein is different from theterminology in U.S. Provisional Application Serial No. 60/107,911 andU.S. Provisional Application Serial No. 60/149,332 upon which thisapplication claims priority in part. As described herein, the term“human mox1” refers to a protein comprising an amino acid sequence asset forth in SEQ ID NO:2, or a fragment thereof, and encoded by thenucleotide sequence as set forth in SEQ ID NO:1, or a fragment thereof.As described herein, the term “human mox2” refers to a proteincomprising an amino acid sequence as set forth in SEQ ID NO:4, or afragment thereof, and encoded by the nucleotide sequence as set forth inSEQ ID NO:3, or a fragment thereof. As described herein, the term “humanduox1” refers to a protein comprising an amino acid sequence as setforth in SEQ ID NO:46, or a fragment thereof, and encoded by thenucleotide sequence as set forth in SEQ ID NO:45, or a fragment thereof.As described herein, the term “human duox2” refers to a proteincomprising an amino acid sequence as set forth in SEQ ID NO:48, or afragment thereof, and encoded by the nucleotide sequence as set forth inSEQ ID NO:47, or a fragment thereof.

Construction of the Recombinant Gene

The desired gene is ligated into a transfer vector, such as pcDNA3, andthe recombinants are used to transform host cells such as Cos-7 cells.It is to be understood that different transfer vectors, host cells, andtransfection methods may be employed as commonly known to one ofordinary skill in the art. Six desired genes for use in transfection areshown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:22, SEQ ID NO:41 SEQ IDNO:45 and SEQ ID NO:47. For example, lipofectamine-mediated transfectionand in vivo homologous recombination was used to introduce the mox1 geneinto NIH 3T3 cells.

The synthetic gene is cloned and the recombinant construct containingmox or duox gene is produced and grown in confluent monolayer culturesof a Cos-7 cell line. The expressed recombinant protein is thenpurified, preferably using affinity chromatography techniques, and itspurity and specificity determined by known methods.

A variety of expression systems may be employed for expression of therecombinant protein. Such expression methods include, but are notlimited to the following: bacterial expression systems, including thoseutilizing E. coli and Bacillus subtilis; virus systems; yeast expressionsystems; cultured insect and mammalian cells; and other expressionsystems known to one of ordinary skill in the art.

Transfection of Cells

It is to be understood that the vectors of the present invention may betransfected into any desired cell or cell line. Both in vivo and invitro transfection of cells are contemplated as part of the presentinvention. Preferred cells for transfection include but are not limitedto the following: fibroblasts (possibly to enhance wound healing andskin formation), granulocytes (possible benefit to increase function ina compromised immune system as seen in AIDS, and aplastic anemia),muscle cells, neuroblasts, stem cells, bone marrow cells, osteoblasts, Blymphocytes, and T lymphocytes.

Cells may be transfected with a variety of methods known to one ofordinary skill in the art and include but are not limited to thefollowing: electroporation, gene gun, calcium phosphate, lipofectamine,and fugene, as well as adenoviral transfection systems.

Host cells transfected with the nucleic acids represented in SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:22, SEQ ID NO:41 SEQ ID NO:45 and SEQ IDNO:47, or fragments thereof, are used to express the proteins SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:21, SEQ ID NO:42, SEQ ID NO:46 and SEQ IDNO:48, respectively, or fragments thereof.

These expressed proteins are used to raise antibodies. These antibodiesmay be used for a variety of applications including but not limited toimmunotherapy against cancers expressing one of the mox or duoxproteins, and for detection, localization and measurement of theproteins shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:21, SEQ ID NO:42,SEQ ID NO:46 or SEQ ID NO:48 or fragments thereof.

Purification and Characterization of the Expressed Protein

The proteins of the present invention can be expressed as a fusionprotein with a poly histidine component, such as a hexa histidine, andpurified by binding to a metal affinity column using nickel or cobaltaffinity matrices. The protein can also be expressed as a fusion proteinwith glutathione S-transferase and purified by affinity chromatographyusing a glutathione agarose matrix. The protein can also be purified byimmunoaffinity chromatography by expressing it as a fusion protein, forexample with hemagglutinin antigen. The expressed or naturally occurringprotein can also be purified by conventional chromatographic andpurification methods which include anion and cation exchangechromatography, gel exclusion chromatography, hydroxylapatitechromatography, dye binding chromatography, ammonium sulfateprecipitation, precipitation in organic solvents or other techniquescommonly known to one of skill in the art.

Methods of Assessing Activity of Expressed Proteins

Different methods are available for assessing the activity of theexpressed proteins of the present invention, including but not limitedto the proteins represented as SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:21,SEQ ID NO:42, SEQ ID NO:46 or SEQ ID NO:48 substituted analogs thereof,and fragments thereof.

1. Assays of the Holoprotein and Fragments thereof for SuperoxideGeneration

A. General Considerations

These assays are useful in assessing efficacy of drugs designed tomodulate the activity of the enzymes of the present invention. Theholoprotein may be expressed in COS-7 cells, NIH 3T3 cells, insect cells(using baculoviral technology) or other cells using methods known to oneof skill in the art. Membrane fractions or purified protein are used forthe assay. The assay may require or be augmented by other cellularproteins such as p47phox, p67phox, and Rac1, as well as potentiallyother unidentified factors (e.g., kinases or other regulatory proteins).

B. Cytochrome c Reduction

NADPH or NADH is used as the reducing substrate, in a concentration ofabout 100 μM. Reduction of cytochrome c is monitoredspectrophotometrically by the increase in absorbance at 550 nm, assumingan extinction coefficient of 21 mM⁻¹ cm⁻¹. The assay is performed in theabsence and presence of about 10 μg superoxide dismutase. Thesuperoxide-dependent reduction is defined as cytochrome c reduction inthe absence of superoxide dismutase minus that in the presence ofsuperoxide dismutase (Uhlinger et al. (1991) J. Biol. Chem. 266,20990-20997). Acetylated cytochrome c may also be used, since thereduction of acetylated cytochrome c is thought to be exclusively viasuperoxide.

C. Nitroblue Tetrazolium Reduction

For nitroblue tetrazolium (NBT) reduction, the same general protocol isused, except that NBT is used in place of cytochrome c. In general,about 1 mL of filtered 0.25% nitrotetrazolium blue (Sigma, St. Louis,Mo.) is added in Hanks buffer without or with about 600 Units ofsuperoxide dismutase (Sigma) and samples are incubated at approximately37° C. The oxidized NBT is clear, while the reduced NBT is blue andinsoluble. The insoluble product is collected by centrifugation, and thepellet is re-suspended in about 1 mL of pyridine (Sigma) and heated forabout 10 minutes at 100° C. to solubilize the reduced NBT. Theconcentration of reduced NBT is determined by measuring the absorbanceat 510 nm, using an extinction coefficient of 11,000 M⁻¹ cm⁻¹. Untreatedwells are used to determine cell number.

D. Luminescence

Superoxide generation may also be monitored with a chemiluminescencedetection system utilizing lucigenin (bis-N-methylacridinium nitrate,Sigma, St. Louis, Mo.). The sample is mixed with about 100 μM NADPH(Sigma, St. Louis, Mo.) and 10 μM lucigenin (Sigma, St. Louis, Mo.) in avolume of about 150 μL Hanks solution. Luminescence is monitored in a96-well plate using a LumiCounter (Packard, Downers Grove, Ill.) for 0.5second per reading at approximately 1 minute intervals for a total ofabout 5 minutes; the highest stable value in each data set is used forcomparisons. As above, superoxide dismutase is added to some samples toprove that the luminescence arises from superoxide. A buffer blank issubtracted from each reading (Ushio-Fukai et al. (1996) J. Biol. Chem.271, 23317-23321).

E. Assays in Intact Cells

Assays for superoxide generation may be performed using intact cells,for example, the mox-transfected NIH 3T3 cells. In principle, any of theabove assays can be used to evaluate superoxide generation using intactcells, for example, the mox-transfected NIH 3T3 cells. NBT reduction isa preferred assay method.

2. Assays of Truncated Proteins Comprised of Approximately theC-terminal 265 Amino Acid Residues

While not wanting to be bound by the following statement, the truncatedprotein comprised of approximately the C-terminal 265 amino acidresidues is not expected to generate superoxide, and therefore,superoxide dismutase is not added in assays of the truncated protein.Basically, a similar assay is established and thesuperoxide-independent. reduction of NBT, cytochrome c,dichlorophenolindophenol, ferricyanide, or another redox-active dye isexamined.

Nucleotides and Nucleic Acid Probes

The nucleotide sequences SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:22, SEQ IDNO:41 SEQ ID NO:45 and SEQ ID NO:47, as well as fragments thereof andPCR primers therefor, may be used, respectively, for localization,detection and measurement of nucleic acids related to SEQ ID NO:1, SEQID NO:3, SEQ ID NO:22, SEQ ID NO:41 SEQ ID NO:45 and SEQ ID NO:47, aswell as fragments thereof. The nucleotide sequences SEQ ID NO:1 and SEQID NO:3 are also called the human mox1 gene and the human mox2 gene inthis application. SEQ ID NO:22 is also known as the rat mox1 gene inthis application. SEQ ID NO:41 is also known as the rat mox1B gene inthis application. SEQ ID NO:45 is also known as the human duox1 gene inthis application. SEQ ID NO:47 is also known as the human duox2 gene inthis application.

The nucleotide sequences SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:22, SEQ IDNO:41 SEQ ID NO:45 and SEQ ID NO:47, as well as fragments thereof, maybe used to create probes to isolate larger nucleotide sequencescontaining the nucleotide sequences SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:22, SEQ ID NO:41 SEQ ID NO:45 and SEQ ID NO:47, respectively. Thenucleotide sequences SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:22, SEQ IDNO:41 SEQ ID NO:45 and SEQ ID NO:47, as well as fragments thereof, mayalso be used to create probes to identify and isolate mox and duoxproteins in other species.

The nucleic acids described herein include messenger RNA coding forproduction of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:21, SEQ ID NO:42, SEQID NO:46, SEQ ID NO:48 and fragments thereof. Such nucleic acids includebut are not limited to cDNA probes. These probes may be labeled in avariety of ways known to one of ordinary skill in the art. Such methodsinclude but are not limited to isotopic and non-isotopic labeling. Theseprobes may be used for in situ hybridization for localization of nucleicacids such as mRNA encoding for SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:21,SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:48 and fragments thereof.Localization may be performed using in situ hybridization at bothultrastructural and light microscopic levels of resolution usingtechniques known to one of ordinary skill in the art.

These probes may also be employed to detect and quantitate nucleic acidsand mRNA levels using techniques known to one of ordinary skill in theart including but not limited to solution hybridization.

Antibody Production

The proteins shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:21, SEQ IDNO:42, SEQ ID NO:46, SEQ ID NO:48, or fragments thereof, are combinedwith a pharmaceutically acceptable carrier or vehicle to produce apharmaceutical composition and administered to animals for theproduction of polyclonal antibodies using methods known to one ofordinary skill in the art. The preferred animals for antibody productionare rabbits and mice. Other animals may be employed for immunizationwith these proteins or fragments thereof. Such animals include, but arenot limited to the following; sheep, horses, pigs, donkeys, cows,monkeys and rodents such as guinea pigs and rats.

The terms “pharmaceutically acceptable carrier or pharmaceuticallyacceptable vehicle” are used herein to mean any liquid including but notlimited to water or saline, oil, gel, salve, solvent, diluent, fluidointment base, liposome, micelle, giant micelle, and the like, which issuitable for use in contact with living animal or human tissue withoutcausing adverse physiological responses, and which does not interactwith the other components of the composition in a deleterious manner.

The pharmaceutical compositions may conveniently be presented in unitdosage form and may be prepared by conventional pharmaceuticaltechniques. Such techniques include the step of bringing intoassociation the active ingredient and the pharmaceutical carrier(s) orexcipient(s). In general, the formulations are prepared by uniformly andintimately bringing into association the active ingredient with liquidcarriers.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example, sealed ampules and vials, and may be stored ina freeze-dried (lyophilized) condition requiring only the addition ofthe sterile liquid carrier, for example, water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletscommonly used by one of ordinary skill in the art.

Preferred unit dosage formulations are those containing a dose or unit,or an appropriate fraction thereof, of the administered ingredient. Itshould be understood that in addition to the ingredients, particularlymentioned above, the formulations of the present invention may includeother agents commonly used by one of ordinary skill in the art.

The pharmaceutical composition may be administered through differentroutes, such as oral, including buccal and sublingual, rectal,parenteral, aerosol, nasal, intramuscular, subcutaneous, intradermal,and topical. The pharmaceutical composition of the present invention maybe administered in different forms, including but not limited tosolutions, emulsions and suspensions, microspheres, particles,microparticles, nanoparticles, and liposomes. It is expected that fromabout 1 to 7 dosages may be required per immunization regimen. Initialinjections may range from about 0.1 μg to 1 mg, with a preferred rangeof about 1 μg to 800 μg, and a more preferred range of fromapproximately 25 μg to 500 μg. Booster injections may range from 0.1 μgto 1 mg, with a preferred range of approximately 1 μg to 800 μg, and amore preferred range of about 10 μg to 500 μg.

The volume of administration will vary depending on the route ofadministration and the size of the recipient. For example, intramuscularinjections may range from about 0.1 ml to 1.0 ml.

The pharmaceutical composition may be stored at temperatures of fromabout 4° C. to −100° C. The pharmaceutical composition may also bestored in a lyophilized state at different temperatures including roomtemperature. The pharmaceutical composition may be sterilized throughconventional means known to one of ordinary skill in the art. Such meansinclude, but are not limited to filtration, radiation and heat. Thepharmaceutical composition of the present invention may also be combinedwith bacteriostatic agents, such as thimerosal, to inhibit bacterialgrowth.

Adjuvants

A variety of adjuvants known to one of ordinary skill in the art may beadministered in conjunction with the protein in the pharmaceuticalcomposition. Such adjuvants include, but are not limited to thefollowing: polymers, co-polymers such aspolyoxyethylene-polyoxypropylene copolymers, including blockco-polymers; polymer P1005; Freund's complete adjuvant (for animals);Freund's incomplete adjuvant; sorbitan monooleate; squalene; CRL-8300adjuvant; alum; QS 21, muramyl dipeptide; trehalose; bacterial extracts,including mycobacterial extracts; detoxified endotoxins; membranelipids; or combinations thereof.

Monoclonal antibodies can be produced using hybridoma technology inaccordance with methods well known to those skilled in the art. Theantibodies are useful as research or diagnostic reagents or can be usedfor passive immunization. The composition may optionally contain anadjuvant.

The polyclonal and monoclonal antibodies useful as research ordiagnostic reagents may be employed for detection and measurement of SEQID NO:2, SEQ ID NO:4, SEQ ID NO:21, SEQ ID NO:42, SEQ ID NO:46, SEQ IDNO:48 and fragments thereof. Such antibodies may be used to detect theseproteins in a biological sample, including but not limited to samplessuch as cells, cellular extracts, tissues, tissue extracts, biopsies,tumors, and biological fluids. Such detection capability is useful fordetection of disease related to these proteins to facilitate diagnosisand prognosis and to suggest possible treatment alternatives.

Detection may be achieved through the use of inmmunocytochemistry,ELISA, radioimmunoassay or other assays as commonly known to one ofordinary skill in the art. The mox1, mox2, duox1 and duox2 proteins, orfragments thereof, may be labeled through commonly known approaches,including but not limited to the following: radiolabeling, dyes,magnetic particles, biotin-avidin, fluorescent molecules,chemiluminescent molecules and systems, ferritin, colloidal gold, andother methods known to one of skill in the art of labeling proteins.

Administration of Antibodies

The antibodies directed to the proteins shown as SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:21, SEQ ID NO:42, SEQ ID NO:46 or SEQ ID NO:48, ordirected to fragments thereof, may also be administered directly tohumans and animals in a passive immunization paradigm. Antibodiesdirected to extracellular portions of SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:21, SEQ ID NO:42, SEQ ID NO:46 or SEQ ID NO:48 bind to theseextracellular epitopes. Attachment of labels to these antibodiesfacilitates localization and visualization of sites of binding.Attachment of molecules such as ricin or other cytotoxins to theseantibodies helps to selectively damage or kill cells expressing SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:21, SEQ ID NO:42, SEQ ID NO:46, SEQ IDNO:48 or fragments thereof.

Kits

The present invention includes kits useful with the antibodies, nucleicacids, nucleic acid probes, labeled antibodies, labeled proteins orfragments thereof for detection, localization and measurement of SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:21, SEQ ID NO:22,SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47,SEQ ID NO:48 or combinations and fragments thereof.

Kits may be used for immunocytochemistry, in situ hybridization,solution hybridization, radioimmunoassay, ELISA, Western blots,quantitative PCR, and other assays for the detection, localization andmeasurement of these nucleic acids, proteins or fragments thereof usingtechniques known to one of skill in the art.

The nucleotide sequences shown in SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:22, SEQ ID NO:41 SEQ ID NO:45, SEQ ID NO:47, or fragments thereof,may also be used under high stringency conditions to detect alternatelyspliced messages related to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:22, SEQID NO:41 SEQ ID NO:45, SEQ ID NO:47 or fragments thereof, respectively.As discussed in one of the Examples, rat mox1 protein (SEQ ID NO: 21) issimilar to mouse gp91 protein (SEQ ID NO: 38), whereas rat mox1B protein(SEQ ID NO:42) is similar to human gp91 protein (SEQ ID NO:12). Thisobservation suggests that other isoforms of mouse and human gp91 mayexist. In addition, another subtype of human mox1, similar to rat mox1B(SEQ ID NO:42), also exists. The presence of two isoforms of rat mox1protein in vascular smooth muscle may have important physiologicalconsequences and biomedical applications. For example, the two isoformsmay have different biological activities, different tissue distributionsand may be regulated differently in physiological and/or pathologicalconditions. The fact that mox1B (SEQ ID NO:42) was isolated from cellsexposed to angiotensin II, known to promote oxidative stress andvascular growth, suggests that it may be upregulated by this hormone andmay be overexpressed in disease. Therefore, the diagnostic kits of thepresent invention can measure the relative expression of the two moxisoforms. The diagnostic kits may also measure or detect the relativeexpression of the mox proteins described herein (i.e. human mox1 and/orhuman mox2) and duox proteins described herein (i.e. human duox1 and/orhuman duox2).

Fragments of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:22, SEQ ID NO:41 SEQ IDNO:45 and SEQ ID NO:47 containing the relevant hybridizing sequence canbe synthesized onto the surface of a chip array. RNA samples, e.g., fromtumors, are then fluorescently tagged and hybridized onto the chip fordetection. This approach may be used diagnostically to characterizetumor types and to tailor treatments and/or provide prognosticinformation. Such prognostic information may have predictive valueconcerning disease progression and life span, and may also affect choiceof therapy.

The present invention is further illustrated by the following examples,which are not to be construed in any way as imposing limitations uponthe scope thereof. On the contrary, it is to be clearly understood thatresort may be had to various other embodiments, modifications, andequivalents thereof, which, after reading the description herein, maysuggest themselves to those skilled in the art without departing fromthe spirit of the present invention.

EXAMPLE 1 Sequence Analysis and Cloning of the Human mox1 cDNA (SEQ IDNO:1) Encoding for Production of the Human mox1 Protein p65mox (SEQ IDNO:2)

Using gp91 phox as a query sequence, a 334 base sequenced portion ofexpressed sequence tag (EST) 176696 (GenBank Accession number AA305700)showed 68.8% sequence identity at the predicted amino acid level withhuman (h) gp91phox. The bacterial strain number 129134 containing theEST sequence in the pBluescript SK⁻ vector, was purchased from AmericanTissue Type Culture Collection (ATCC, Rockville, Md.). The EST sequencewas originally cloned from a Caco-2 human colon carcinoma cell line. TheEST176696 DNA was further sequenced using the T7 and T3 vector promotersand primers designed to match the known 3′ sequence. Internal primersused for sequencing were as follows: 5′-AAC AAG CGT GGC TTC AGC ATG-3′SEQ ID NO:5 (251S, numbering is based on the nucleotides from the 5′ endof EST176696, and S indicates the sense direction), 5′-AGC AAT ATT GTTGGT CAT-3′ SEQ ID NO:6 (336S), 5′-GAC TTG ACA GAA AAT CTA TAA GGG-3′ SEQID NO:7 (393S), 5′-TTG TAC CAG ATG GAT TTC AA-3′ SEQ ID NO:8 (673A, Aindicates the antisense direction), 5′-CAG GTC TGA AAC AGA AAA CCT-3′SEQ ID NO:9 (829S), 5′-ATG AAT TCT CAT TAA TTA TTC AAT AAA-3′ SEQ IDNO:10 (1455A). The coding sequence in EST176696 showed homology to a 250amino acid stretch corresponding to the N-terminal 44% of humangp91phox, and contained a stop codon corresponding to the location inhuman gp91 phox. 5′ Rapid amplification of cDNA ends (RACE) was carriedout using a human colon cDNA library and Marathon cDNA Amplification Kit(ClonTech) using 5′-ATC TCA AAA GAC TCT GCA CA-3′ SEQ ID NO:11 (41A) asan internal gene-specific primer (Frohman et al. (1988) Proc. Natl.Acad. Sci. USA 85, 8998-9002). 5′ RACE resulted in a 1.1 kb fragmentrepresenting the complete 5′ sequence, based on homology with gp91phox.Reamplification was performed with primers spanning the putative startand stop codons, using the 1.1 kb 5′ RACE product and pSK-EST176696 forprimer design. The amplified 1.7 kb fragment was TA cloned into thePCR2.1 vector (Invitrogen, Carlsbad, Calif.). This recombinant vector isreferred to as PCR-mox.

FIG. 1(a-d) presents a comparison of the present amino acid sequences ofhuman, bovine and murine gp91 phox with the human and rat mox1 proteinsof the present invention and the human duox2 protein of the presentinvention. Also shown are the amino acid sequences for related plantenzyme proteins.

The encoded hp65mox (“mox” referring to mitogenic oxidase and “65”referring to its predicted molecular weight) is listed as SEQ ID NO:2.h-gp91phox (SEQ ID NO:12) and SEQ ID NO:2 differ in length by 3 residuesand are 70% identical in their amino acid sequence. h-gp91phox and SEQID NO:2 show a greater percentage identity in the C-terminal half of themolecule which contains the putative NADPH and FAD binding sites, andthere are several relatively long stretches of complete identity withinthis region.

A dendrogram (FIG. 2) comparing the amino acid sequences of mouse andhuman gp91phox with that of mox1 SEQ ID NO:2 shows that the latterprobably represents a distinct isoform of gp91phox. Two plant homologsof cytochrome b₅₅₈ large subunit are also indicated and represent moredistant relatives of the human sequences. Human (and rat mox1 describedmore fully below) lack asparagine-linked glycosylation sites, which areseen in the highly glycosylated human and mouse gp91phox. Additionally,the hydropathy profiles of human gp91phox and mox1 are nearly identicaland include five very hydrophobic stretches in the amino-terminal halfof the molecules which are predicted to be membrane-spanning regions.

EXAMPLE 2 Expression of Mox1

Human multiple tissue northern (MTN) Blot I and Human MTN Blot IV(ClonTech) membranes were hybridized with the putative coding region ofthe PCR-mox vector at 68° C. for several hours. The mox coding regionwas labeled by random priming with [α-³²P]dCTP (10 μCi) using thePrime-It II kit (Stratagene). For analysis of mox1 expression in celllines, total RNA was prepared from 10⁶ cells using the High Pure RNAIsolation Kit (Boehringer Mannheim) or RNeasy kit (Quiagen). Total RNA(10-20 μg) was separated on a 1% agarose formaldehyde mini-gel andtransferred to a Nytran filter (Biorad) and immobilized by ultravioletcross-linking.

Northern blotting revealed that the major location of MRNA coding forthe mox1 protein was colon. The message was also detected in prostateand uterus. The human colon-carcinoma cell line, Caco-2, also expressedlarge quantities of mox1 message. Northern blotting of mRNA from rataortic smooth muscle cells also showed strong hybridization, whichincreased roughly two-fold within 12 hours after treatment withplatelet-derived growth factor. This increase in the expression of ratmox1 is consistent with the idea that mox1 contributes to thegrowth-stimulatory effects of PDGF.

EXAMPLE 3 Transfection of NIH3T3 Cells with SEQ ID NO:1

The nucleotide sequence (SEQ ID NO:1) encoding for production of themox1 protein (SEQ ID NO:2) was subcloned into the Not1 site of thepEF-PAC vector (obtained from Mary Dinauer, Indiana University MedicalSchool, Indianapolis, Ind.) which has a puromycin resistance gene.Transfection was carried out as described in Sambrook et al., MolecularCloning, A Laboratory Manual, Volumes 1-3, 2nd edition, Cold SpringHarbor Laboratory Press, N.Y., 1989. The SEQ ID NO:1 in pEF-PAC and theempty vector were separately transfected into NIH 3T3 cells using Fugene6 (Boeringer Mannheim). About 2×10⁶ cells maintained in DMEM containing10% calf serum were transfected with 10 μg of DNA. After 2 days, cellswere split and selected in the same medium containing 1 mg/ml puromycin.Colonies that survived in selection media for 10 to 14 days weresubcultured continuously in the presence of puromycin.

Transfected cells exhibited a “transformed”-like morphology, similar tothat seen with (V12)Ras-transfected cells, characterized by longspindle-like cells. The parent NIH 3T3 cells or cells transfected withthe empty vector showed a normal fibroblast-like morphology.

EXAMPLE 4 Expression of Mox1 (SEQ ID NO:1) in Transfected NIH3T3 Cells

To verify the expression of mox1 mRNA after transfection, RT-PCR andNorthern blotting were performed. Total RNAs were prepared from 10⁶cells using the High Pure RNA Isolation Kit (Boeringer Mannheim) orRNeasy kit (Qiagen). cDNAs for each colony were prepared from 1-2 μg oftotal RNA using Advantage RT-PCR Kit (ClonTech). PCR amplification wasperformed using primers, 5′-TTG GCT AAA TCC CAT CCA-3′ SEQ ID NO:13(NN459S, numbering containing NN indicates numbering from the startcodon of mox1) and 5′-TGC ATG ACC AAC AAT ATT GCT G-3′ SEQ ID NO:14(NN1435A). For Northern blotting, 10-20 μg of total RNA was separated ona 1% agarose formaldehyde gel and transferred to a nylon filter. Afterultraviolet (UV) cross-linking, filters were used for Northern blottingassay as described in Example 2.

Colonies expressing large amounts of mox1 mRNA were chosen for furtheranalysis. The expression of mRNA for glyceraldehyde 3 phosphatedehydrogenase in the various cell lines was normal.

EXAMPLE 5 Colony Formation on Soft Agar

10⁵ to 10³ cells stably transfected with human mox1 gene SEQ ID NO:1 andwith empty vector were prepared in 0.3% warm (40° C.) agar solutioncontaining DMEM and 10% calf serum. Cells were distributed onto ahardened 0.6% agar plate prepared with DMEM and 10% calf serum. Afterthree weeks in culture (37° C., 5% CO₂) colony formation was observed bymicroscopy.

Cells which were stably transfected with the empty vector and culturedin soft agar for 3 weeks as above did not display anchorage independentgrowth. In contrast, NIH 3T3 cells which had been stably transfectedwith the mox1 (SEQ ID NO:1) and cultured for 3 weeks in soft agardemonstrated anchorage independent growth of colonies.

EXAMPLE 6 NADPH-Dependent Superoxide Generation Assay

In one embodiment of the present invention, NIH 3T3 cells stablytransfected with the human mox1 gene (SEQ ID NO:1) were analyzed forsuperoxide generation using the lucigenin (Bis-N-methylacridiniumluminescence assay (Sigma, St. Louis, Mo., Li et al. (1998) J. Biol.Chem. 273, 2015-2023). Cells were washed with cold HANKS' solution andhomogenized on ice in HANKS' buffer containing 15% sucrose using aDounce homogenizer. Cell lysates were frozen immediately in a dryice/ethanol bath. For the assay, 30 μg of cell lysate was mixed with 200μM NADPH and 500 μM lucigenin. Luminescence was monitored using aLumiCounter (Packard) at three successive one minute intervals and thehighest value was used for comparison. Protein concentration wasdetermined by the Bradford method.

Superoxide generation was monitored in lysates from some of the stablytransfected cell lines and was compared with superoxide generation bythe untransfected NIH 3T3 cell lysates. The results are shown in Table4. Cell lines 26, 27, and 28 gave the highest degree of morphologicalchanges by microscopic examination corresponding to the highest degreeof superoxide generation. The luminescent signal was inhibited bysuperoxide dismutase and the general flavoprotein inhibitor diphenyleneiodonium, but was unaffected by added recombinant human p47phox, p67phoxand Rac1.(GTP-γS), which are essential cytosolic factors for thephagocyte respiratory-burst oxidase.

TABLE 4 Superoxide Generation Cell Line Name (RLU) Control(untransfected) 6045 mox1-26 17027 mox1-27 14670 mox1-28 18411 mox1-655431 mox1-615 11331 mox1-+3 8645 mox1-+10 5425 mox1-pcc16 8050

In an alternate and preferred embodiment of the present invention, cellsthat had been stably transfected with mox1 (YA28) or with empty vector(NEF2) were grown in 10 cm tissue culture plates in medium containingDMEM, 10% calf serum, 100 units/ml penicillin, 100 μg/ml streptomycin,and 1 μg/ml puromycin to approximately 80% confluency. Cells (fivetissue culture plates of each cell type) were washed briefly with 5 mlphosphate buffered saline (PBS) then dissociated from the plates withPBS containing 5 mM EDTA. Cells were pelleted by centrifuging briefly at1000×g.

To permeabilize the cells, freeze thaw lysis was carried out and thiswas followed by passage of the cell material through a small boreneedle. The supernatant was removed and the cells were frozen on dry icefor 15 minutes. After cells were thawed, 200 μl lysis buffer (HANKS'Buffered Salt Solution—HBBS) containing a mixture of protease inhibitorsfrom Sigma (Catalog #P2714) was added. Cells on ice were passed throughan 18 guage needle 10 times and 200 μl of HBSS buffer containing 34%sucrose was added to yield a final concentration of 17% sucrose. Sucroseappeared to enhance stability upon storage. The combination offreeze-thawing and passage through a needle results in lysis ofessentially all of the cells, and this material is referred to as the“cell lysate.”

The cell lysates were assayed for protein concentration using the BioRadprotein assay system. Cell lysates were assayed for NADPH-dependentchemiluminescence by combining HBSS buffer, arachidonic acid, and 0.01-1μg protein in assay plates (96 well plastic plates). The reaction wasinitiated by adding 1.5 mM NADPH and 75 μM lucigenin to the assay mix togive a final concentration of 200 μM NADPH and 10 μM lucigenin, and thechemiluminescence was monitored immediately. The final assay volume as150 μl. The optimal arachidonic acid concentration was between 50-100μM. A Packard Lumicount luminometer was used to measurechemiluminescence of the reaction between lucigenin and superoxide at37° C. The plate was monitored continuously for 60 minutes and themaximal relative luminescence unit (RLU) value for each sample was usedfor the graph.

FIG. 3 shows the RLU at various concentrations of cell lysates frommox1-transfected (YA28) and vector control (NEF2) cells. The presence ofNaCl or KCl within a concentration range of 50-150 μM is important foroptimal activity. MgCl₂ (1-5 mM) further enhanced activity by about2-fold. This cell-free assay for mox1 NADPH-oxidase activity is usefulfor screening modulators (inhibitors or stimulators) of the mox1 enzyme.The assay may also be used to detect mox and duox NADPH-oxidase activityin general and to screen for modulators (inhibitors or stimulators) ofthe mox and duox family of enzymes.

EXAMPLE 7 Nitro Blue Tetrazolium Reduction by Superoxide Generated byNIH 3T3 cells Transfected with the Mox1 cDNA (SEQ ID NO:1)

Superoxide generation by intact cells was monitored by using superoxidedismutase-sensitive reduction of nitroblue tetrazolium. NEF2 (vectoralone control), YA26 (mox1 (SEQ ID NO:1)-transfected) and YA28 (mox1(SEQ ID NO:1)-transfected) cells were plated in six well plates at500,000 cells per well. About 24 hours later, medium was removed fromcells and the cells were washed once with 1 mL Hanks solution (Sigma,St. Louis, Mo.). About 1 mL of filtered 0.25% Nitro blue tetrazolium(NBT, Sigma) was added in Hanks without or with 600 units of superoxidedismutase (Sigma) and cells were incubated at 37° C. in the presence of5% CO₂. After 8 minutes the cells were scraped and pelleted at more than10,000 g. The pellet was re-suspended in 1 mL of pyridine (Sigma) andheated for 10 minutes at 100° C. to solubilize the reduced NBT. Theconcentration of reduced NBT was determined by measuring the absorbanceat 510 nm, using an extinction coefficient of 11,000 M⁻¹cm⁻¹. Some wellswere untreated and used to determine cell number.

The data are presented in Table 5 and FIG. 4 and indicate that the mox1(SEQ ID NO:1)-transfected cells generated significant quantities ofsuperoxide.

TABLE 5 NBT Reduction (nmols/10⁶ cells) −SOD +SOD vector control cells2.5 ± 0.5 2.1 ± 0.5 YA26 (mox1) cells 6.4 ± 0.2 3.4 ± 0.1 YA28 (mox1)cells 5.2 ± 0.6 3.4 ± 0.3 −SOD, and +SOD mean in the absence or presenceof added superoxide dismutase, respectively.

Because superoxide dismutase is not likely to penetrate cells,superoxide must be generated extracellularly. The amount of superoxidegenerated by these cells is about 5-10% of that generated by activatedhuman neutrophils.

EXAMPLE 8 Modification of Intracellular Components in Mox1 TransfectedCells

To test whether superoxide generated by mox1 can affect intracellular“targets,” aconitase activity in control and mox-transfected cell lineswas monitored as described in Suh et al. (1999) Nature 401, 79-82.Aconitase contains a four-iron-sulphur cluster that is highlysusceptible to modification by superoxide, resulting in a loss ofactivity, and has been used as a reporter of intra-cellular superoxidegeneration. Acotinase activity was determined as described in Gardner etal. (1995) J. Biol. Chem. 270, 13399-13405. Acotinase activity wassignificantly diminished in all three mox-transfected cell linesdesignated YA26, YA28 and YA212 as compared to the transfected control(FIG. 5). Approximately 50% of the aconitase in these cells ismitochondrial, based on differential centrifugation, and the cytosolicand mitochondrial forms were both affected. Control cytosolic andmitochondrial enzymes that do not contain iron-sulfur centres were notaffected. Superoxide generated in mox1-transfected cells is thereforecapable of reacting with and modifying intracellular components.

EXAMPLE 9 Tumor Generation in Nude Mice Receiving Cells Transfected withthe Human mox1 cDNA (SEQ ID NO:1)

About 2×10⁶ NIH 3T3 cells (either mox1-transfected with SEQ ID NO:1 orcells transfected using empty vector) were injected subdermally into thelateral aspect of the neck of 4-5 week old nude mice. Three to six micewere injected for each of three mox1-transfected cell lines, and 3 micewere injected with the cells transfected with empty vector (control).After 2 to 3 weeks, mice were sacrificed. The tumors were fixed in 10%formalin and characterized by histological analysis. Tumors averaged1.5×1×1 cm in size and showed histology typical of sarcoma type tumors.In addition, tumors appeared to be highly vascularized with superficialcapillaries. Eleven of twelve mice injected with mox1 gene-transfectedcells developed tumors, while none of the three control animalsdeveloped tumors.

In another study, 15 mice were injected with mox1-transfected NIH 3T3cells. Of the 15 mice injected, 14 showed large tumors within 17 days ofinjection, and tumors showed expression of mox1 mRNA. Histologically,the tumors resembled fibrosarcomas and were similar to ras-inducedtumors. Thus, ras and mox1 were similarly potent in their ability toinduce tumorigenicity of NIH 3T3 cells in athymic mice.

EXAMPLE 10 Demonstration of the Role of Mox1 in Non-Cancerous Growth

A role in normal growth was demonstrated in rat aortic vascularsmooth-muscle cells by using antisense to rat mox1. Transfection withthe antisense DNA resulted in a decrease in both superoxide generationand serum-dependent growth. Mox1 is therefore implicated in normalgrowth in this cell type.

EXAMPLE 11 Expression of Human Mox1 Protein (SEQ ID NO:2) in aBaculovirus Expression System

SEQ ID NO:2 was also expressed in insect cells using recombinantbaculovirus. To establish the p65mox1 expressing virus system, the mox1gene (SEQ ID NO:1) was initially cloned into the pBacPAK8 vector(Clontech, Palo Alto, Calif.) and recombinant baculovirus wasconstructed using standard methods according to manufacturer'sprotocols. Briefly, PCR amplified mox1 DNA was cloned into the KpnI andEcoRI site of the vector. Primers used for PCR amplification were:5′-CAA GGT ACC TCT TGA CCA TGG GAA ACT-3′, SEQ ID NO:15, and 5′-ACG AATTCA AGT AAA TTA CTG AAG ATA C-3′, SEQ ID NO:16. Sf9 insect cells (2×10⁶cells) were infected with 0.5 mg of linearized baculovirus DNA soldunder the trademark BACULOGOLD® (PharMingen, San Diego, Calif.) and 5 mgpBacPAC8-p65mox1 using Transfection Buffers A and B (PharMingen, SanDiego, Calif.). After 5 days, the supernatants containing recombinantviruses were harvested and amplified by infecting fresh sf9 cells for 7days. Amplification was carried out three times and the presence of therecombinant virus containing mox1 DNA was confirmed by PCR using thesame primers. After three times amplification of viruses, plaquepurification was carried out to obtain the high titer viruses.Approximately 2×10⁸ sf9 cells in agar plates were infected for 5 dayswith serial dilutions of virus and were dyed with neutral red for easydetection of virus plaques. Selected virus plaques were extracted andthe presence of the human mox1 DNA was confirmed again by PCR.

EXAMPLE 12 Cloning of a Rat Homolog of p65mox (SEQ ID NO:2)

cDNA clones of p65mox from a rat aortic smooth muscle cell have beenobtained. RT-PCR (reverse transcription polymerase chain reaction) wascarried out as follows: first strand cDNA synthesis was performed usingtotal RNA from rat aortic vascular smooth muscle cells, oligo dT primerand superscript II reverse transcriptase, and followed by incubationwith RNase H. Degenerate PCR primers were designed to anneal toconserved areas in the coding regions of h-mox1 and gp91phox of human(X04011), mouse (U43384) and porcine (SSU02476) origin. Primers were:sense 5′-CCIGTITGTCGIAATCTGCTSTCCTT-3′, SEQ ID NO:17 and antisense5′-TCCCIGCAIAICCAGTAGAARTAGATCTT-3′, SEQ ID NO:18. A major PCR productof the expected 1.1 kb size was purified by agarose electrophoresis andused as template in a second PCR amplification reaction.

An aliquot of the RT-PCR product was blunt-ended, ligated into amodified Litmus 29 vector and used to transform XL10 competent E. coli.Approximately 120 bacterial colonies were screened for the presence of afull-length insert by direct PCR using vector primers and Taqpolymerase. Plasmids were purified from 25 positive colonies and mappedby digestion with Bam HI. Representative plasmids from each digestionpattern were partially sequenced. Five out of 25 clones containednon-specific amplification products and 20 contained identical insertssimilar to human (h)-mox1. One of the latter clones was fully sequencedand found to be 83% identical to h-mox1 over 1060 nucleotides. A 1.1 kbprobe was generated by PCR amplification of the insert of a rat mox1clone with the degenerate primers described above and used to hybridizeto a Northern blot of rat vascular smooth muscle cell RNA. A singleband, migrating between 28S rRNA and 18S rRNA, indicated the presence ofa message with a size compatible to that of human mox-1 (2.6 kb).

To obtain full-length rat mox1, 3′ and 5′ rapid amplification of cDNAends (RACE) reactions were performed as describe above, using thegene-specific primers 5′-TTGGCACAGTCAGTGAGGATGTCTTC-3′, SEQ ID NO:19 and5 ′-CTGTTGGCTTCTACTGTAGCGTTCAAAGTT-3′, SEQ ID NO:20 for 3′ and 5′ RACE,respectively. Single major 1.5 kb and 850 bp products were obtained for3′ and 5′ RACE, respectively. These products were purified by agarosegel eletrophoresis and reamplified with Taq polymerase. Both productswere cloned into the pCR 2.1 vector and used to transformelectrocompetent XL1 blue E. coli. The RACE products were sequenced andnew terminal primers were designed: sense5′-TTCTGAGTAGGTGTGCATTTGAGTGTCATAAAGAC-3′ (SEQ ID NO:43), and antisense5′-TTTTCCGTCAAAATTATAACTTTTTATTTTCTTTTTAT AACACAT-3′ (SEQ ID NO:44). PCRamplification of rat VSMC cDNA was performed using these primers.

A single 2.6 kb product was obtained, ligated into pCR 2.1 and used totransform electrocompetent XL1 blue E. coli. The insert was sequencedwith 12 sense and 14 antisense primers. Its length is 2577 bp (includingprimer sequences), comprising a 1692 bp open reading frame, 127 bp 5′and 758 bp 3′ untranslated regions. The presence of six in-frame stopcodons in the 5′ untranslated region suggests that the full lengthcoding region has been obtained. Consensus polyadenylation sequences arepresent at nucleotides 2201 and 2550. Conceptual translation yields a563 amino acid peptide, one residue shorter than the human deducedsequence. This new amino acid sequence is more similar to human mox1 SEQID NO:3 (82% identity) than to mouse gp91phox SEQ ID NO:38 (55%identity), suggesting that it is indeed rat mox1 (SEQ ID NO:21). Thisrat (r) homolog of p65mox protein is called r-p65mox or p65mox/rat.pepand is shown as SEQ ID NO:21. The nucleotide sequence encoding forr-p65mox is shown as SEQ ID NO:22

EXAMPLE 13 Expression of Rat (r)-p65mox mRNA in Vascular Smooth Muscleand Induction by Angiotensin II, Platelet-Derived Growth Factor (PDGF),and Phorbol Myristic Acid (PMA)

Using the partial cDNA clone from rat, we have examined cultured rataortic smooth muscle cells for expression of message for r-p65mox. Wehave observed the mRNA for r-p65mox in these cells. It has beenpreviously reported (Griendling et al. (1994) Circ. Res. 74, 1141-1148;Fukui et al. (1997) Circ. Res. 80, 45-51; Ushio-Fukai et al. (1996) J.Biol. Chem. 271, 23317-23321) that in vitro or in vivo treatment withangiotensin II (AII) is a growth stimulus for vascular smooth musclecells, and that AII induces increased superoxide generation in thesecells. Platelet-derived growth factor (PDGF) and PMA are proliferativesignals for vascular smooth muscle cells. We observed that the mRNA forr-p65mox was induced approximately 2-3 fold by angiotensin II (100 nM),corresponding to the increased level of superoxide generation. Thus, theincreased superoxide generation in these cells correlates with increasedexpression of the mRNA for this enzyme. The mRNA for r-p65mox alsoincreased 2 or more fold in response to the growth stimulus PDGF (20ng/ml), and 2-3 fold in response to PMA. Quantitation by densitometryrevealed that rat mox1 message was induced nearly 4-fold at the 6 and 12hour time points in response to PDGF, and about 2-fold at the 12 hourtime point in response to AII. 28S RNA was used as a control for RNArecovery.

EXAMPLE 14 Antibodies to Fragments of Human (h)-p65mox (SEQ ID NO:2)

Polyclonal antibodies were raised in rabbits against the C-terminal halfof h-p65mox (residues 233 through 564, SEQ ID NO:23) which is predictedto fold into a cytosolic domain containing FAD and the NADPH or NADHbinding site. This domain was expressed in E. coli as an N-terminalGST-fusion protein and was purified on glutathione agarose by standardmethods. Two antipeptide antibodies were also made against h-p65mox(residues 243-256, referred to as Peptide A, SEQ ID NO:24) and h-65mox(residues 538-551, referred to as Peptide B, SEQ ID NO:25). Peptideswere conjugated to keyhole limpet hemocyanin (KLH) using glutaraldehyde.

Antigens were injected into different rabbits initially in completeFreund's adjuvant, and were boosted 4 times with antigen in incompleteFreund's adjuvant at intervals of every three weeks. Approximately 0.5mg to 1 mg of peptide was administered at each injection. Blood wasdrawn 1 week after each boost and a terminal bleed was carried out 2weeks after the final boost. Antibodies to Peptide A and Peptide B wereaffinity purified by column chromatography through peptide A or peptideB conjugated to Affigel 15 (Bio-Rad, Richmond, Calif.). 10 mg of peptidewas covalently crosslinked to 2 ml of Affigel 15 resin and the gel waswashed with 20 ml of binding buffer (20 mM Hepes/NaOH, pH 7.0, 200 mMNaCl, and 0.5% Triton X-100). The remaining functionalN-hydrosuccinimide was blocked with 100 μl of 1 M ethanolamine. Afterwashing with 20 ml of binding buffer, 5 ml of the antiserum wasincubated with the pep A-conjugated Affigel 15 resin overnight at 4° C.Unbound protein was washed away with 20 ml of binding buffer. Elution ofthe antibodies from the gel was performed with 6 ml of elution buffer(100 mM glycine/HCl, pH 2.5, 200 mM NaCl, and 0.5% Triton X-100). Theeluate was then neutralized by adding 0.9 ml of 1 M Tris/HCl, pH 8.0.The GST-fusion form of truncated p65mox1 protein (residues 233-566, SEQID NO:23) was expressed in E. coli. Samples (20 μg each) were run on 12%SDS-PAGE either before or 1 or 4 hours after induction with 100 μM IPTG(isopropyl β-thiogalactoside).

The extracted proteins were subjected to immunoprobing with affinitypurified antiserum to peptide A at a 1:1000 dilution. The detection ofantigens was performed using an enhanced chemiluminescence kit(Amersham, Buckinghamshire, UK). The affinity purified antibody to mox1(243-256, SEQ ID NO:24) was used at a dilution of 1:1000 in a Westernblot in which a total of 10 μg of protein was added to each lane. Themajor band observed at 4 hours after IPTG induction corresponded to thesize of the GST-mox1 expressed in bacteria containing the pGEX-2T vectorencoding the GST-mox1 fusion protein.

EXAMPLE 15 Presence of an NAD(P)H Oxidase in Ras-Transformed Fibroblasts

A superoxide-generating NADPH oxidase activity was detected inhomogenates from NIH 3T3 cells, and this activity increased about 10-15fold in Ras-transformed NIH 3T3 cells (Table 6). To establish the stableRas-transformed cell lines, the DNA for human Ras encoding an activatingmutation at amino acid number 12 (Valine, referred to as V12-Ras) wassubcloned into BamH1 and EcoR1 sites of pCDNA3 vector which has aneomycin resistance gene. V12-Ras in pCDNA3 and empty vector weretransfected into NIH 3T3 cells using Lipofectamine Plus (Gibco). 2×10⁶cells were maintained with DMEM containing 10% calf serum andtransfected with 1 mg of DNA. After 2-days, cells were split andselected with the same medium but containing 1 mg/ml neomycin. Coloniessurviving in selection media for 10 to 14 days were sub-cultured andcharacterized by immunoblot analysis using antibody against human H-Ras.

The expression of Ras in cells transfected with pcDNA-3 vector alone orin three cell lines transfected with V12-Ras in the same vector wasanalyzed on a Western blot. The three cell lines were named V12-Ras-7,V12-Ras-4, and V12-Ras-8. The expression of V12-Ras varied widely amongthe three cell lines tested. The V12-Ras-4 cell line expressed thehighest level of Ras followed by the V12-Ras-8 cell line. The V12-Ras-7cell line expressed the lowest level of Ras.

Lysates from each of these lines were then prepared and tested for theirability to generate superoxide. For each cell line, cells were washedwith cold HANKS' balanced salt solution (HBSS), collected bycentrifugation, kept on dry-ice for more than 30 min, and disrupted bysuspending in low salt buffer (LSB; 50 mM Tris/HCl, pH 7.5, 1 mM PMSF,and protease cocktail from Sigma) and passing through a syringe needle(18 gauge) ten times. Cell lysates were frozen in dry-ice immediatelyafter determining the protein concentration.

Table 6 shows superoxide generation in the transfected cells measuredusing the lucigenin luminescence assay. For the assay, 5 μg of celllysates were incubated with the reaction mixture containing 10 μMlucigenin (luminescent probe) and 100 μM NADPH (substrate) in thepresence or absence of 100 μM arachidonate in the absence or presence of100 U of superoxide dismutase (SOD) or 1 μM diphenyleneiodonium (DPI).Luminescence of the reaction mixture was monitored for 0.5 second byLumiCounter (Packard) for four times at 3 second intervals. RLU in Table5 refers to relative luminescence units.

As shown in Table 6, the luminescence was partially inhibited bysuperoxide dismutase indicating that the signal was due at least in partto the generation of superoxide. DPI, a known inhibitor of bothneutrophil and non-neutrophil NADPH oxidase activities, completelyinhibited activity. The generation of superoxide correlated with theexpression of Ras in the three cell lines. Thus, oncogenic Ras appearsto induce an NADPH-dependent superoxide generating activity that issimilar to the activity catalyzed by p65mox1.

TABLE 6 RLU/5 μg protein DPI no additions plus SOD plus Vector Control(1) 465 154 48 V12-Ras-7 (2) 1680 578 39 V12-Ras-4 (3) 5975 2128 36V12-Ras-8 (4) 4883 2000 35

EXAMPLE 16 Molecular Cloning of Another Rat mox1 cDNA Called Rat mox1B

A rat cDNA library was screened in an effort to identify new rat moxsequences. The library was constructed in a ZAP express lambda phagevector (Stratagene, La Jolla, Calif.) using RNA isolated from ratvascular smooth muscle cells which had been exposed to 100 nMangiotensin II for 4 hours. The library was screened using standard blothybridization techniques with the rat mox1 probe described previously.Fifteen individual clones were obtained that were characterized by PCRand restriction mapping. Two different types of clones were thusidentified and representatives of each type were sequenced. A clone ofthe first type (representative of 13) was found to be similar to thepreviously identified rat mox1 and was thus named rat mox1B. Clones ofthe second type (representative of 2) were incomplete rat mox sequences.

The length of the rat mox1B nucleotide sequence is 2619 bp and is listedas SEQ ID NO:41. The single longest 1497 bp open reading frameencompasses nucleotides 362 to 1858. The presence of two in-frame stopcodons in the 5′ untranslated region at nucleotides 74 and 257 indicatesthat the full-length coding region has been isolated. Two putativepolyadenylation sites are present at positions 2243 and 2592. Alignmentof the rat mox1 nucleotide sequence (SEQ ID NO:22) and the rat mox1Bnucleotide sequence (SEQ ID NO:41) shows that the two nucleotidessequences are identical except at their 5′ ends, suggesting that theymay represent two alternatively spliced messages from the same gene.Sequence identity starts at nucleotides 269 and 311, for rat mox1 andrat mox1B, respectively.

Conceptual translation of the rat mox1B nucleotide sequence (SEQ IDNO:41) yields a 499 amino acid sequence with a predicted molecularweight of 58 kDa. This amino acid sequence for rat mox1B protein isshown in SEQ ID NO:42. Alignment of the deduced amino acid sequences forrat mox1 (SEQ ID NO:21) and rat mox1B (SEQ ID NO:42) indicates that ratmox1B is identical to rat mox1A, except for a missing stretch of 64residues at the N-terminus. Therefore, rat mox1B appears to be asplicing variant derived from the same gene as rat mox1.

EXAMPLE 17 Sequence Analysis and Cloning of the Human Mox2 cDNA (SEQ IDNO:3) Encoding for Production of the Human Mox2 Protein (SEQ ID NO:4)

Note that the mox2 protein as described herein, was described in U.S.Provisional Application Serial No. 60/149,332 as mox3.

A blast search was carried out using the sequence of mox1 as a querysequence. The sequence identified by this search was a sequence presentin the GenBank database that contains regions of homology with mox1 andgp91phox. The GenBank sequence located in the search was a 90.6 kbsequenced region of human chromosome 6 (6q25.1-26) that was reported asa GenBank direct submission dated Feb. 9, 1999 and given the AccessionNo. AL031773. Sequencing was carried out as part of the human genomesequencing project by S. Palmer, at Sanger Centre, in Hinxton,Cambridgeshire, UK. The GenBank sequence was reported as being similarto “Cytochrome B” and was not reported as having any homology orrelation to a mox protein. The sequence contained a theoretical aminoacid sequence that was derived by computer using an algorithm thatpredicted intron/exon boundaries and coding regions. This predictedregion contained a 545 amino acid sequence that was 56% identical tomox1 and 58% identical to gp91phox.

In the present invention, based on the GenBank genomic sequence and thehomologies described above, several specific primers were designed andused to determine the tissue expression patterns of a novel mox protein,mox2, using Human Multiple Tissue PCR Panels (Clontech, Palo Alto,Calif.). The primers were as follows: Primer 1:5′-CCTGACAGATGTATTTCACTACCCAG-3′ (SEQ ID NO:49); Primer 2:5′-GGATCGGAGTCACTCCCTTCGCTG-3′ (SEQ ID NO:50); Primer 3:5′-CTAGAAGCTCTCCTTGTTGTAATAGA-3′ (SEQ ID NO:51); Primer 4:5′-ATGAACACCTCTGGGGTCAGCTGA-3′ (SEQ ID NO:52). It was determined thatmox2 is expressed primarily in fetal tissues, with highest expression infetal kidney, with expression also seen in fetal liver, fetal lung,fetal brain, fetal spleen and fetal thymus. Among 16 adult tissuestested, mox2 expression was seen in brain, kidney, colon and lung,although levels of expression appeared to be very low.

Additionally, the 5′ RACE (RACE=Rapid Amplification of cDNA Ends) and 3′RACE techniques were used to complete the sequence of the 5′ and 3′regions of mox2. (5′ RACE kit and 3′ RACE kit were from Clontech, PaloAlto, Calif. and are more fully described in Frohman et al. (1988) Proc.Natl. Acad. Sci. USA 85, 8998-9002. The 5′ RACE and 3′-RACE techniqueswere carried out using a human fetal kidney library (Marathon-Ready cDNAlibrary, Cat. #7423-1), using the following specific primers: 5′-RACE:Primer 4: 5′-ATGAACACCTCTGGGGTCAGCTGA-3′ (SEQ ID NO:53); Primer 5:5′-GTCCTCTGCAGCATTGTTCCTCTTA-3′ (SEQ ID NO:54); 3′-RACE: Primer 1:5′-CCTGACAGATGTATTTCACTACCCAG-3′ (SEQ ID NO:55); Primer 2:5′-GGATCGGAGTCACTCCCTTCGCTG-3′ (SEQ ID NO:56). The RACE procedures weresuccessful in completing the 5′ sequence and in confirming the 3′sequence. The complete coding sequence of mox2 is shown in SEQ ID NO:2,while the predicted amino acid sequence of mox2 is shown in SEQ ID NO:4.

In comparing the sequences of the present invention to the predictedcoding regions of the GenBank sequence, the GenBank sequence did notcontain a start codon, appeared to be missing approximately 45 basepairs at the N-terminus, and contained one other major difference in thepredicted coding region which could have been due to inaccurate computerprediction of intron/exon boundaries.

EXAMPLE 18 Sequence Analysis and Partial Cloning of the Human Duox2 cDNA(SEQ ID NO:47) Encoding for Production of the Human Duox2 Protein (SEQID NO:48)

A partial cDNA clone of duox2 was obtained as follows. A 535-baseportion of an expressed sequence tag (EST zc92h03.rl; Genbank accessionno. W52750) from human pancreatic islet was identified using the humangp91phox amino-acid sequence as a query in a Blast search. The bacterialstrain #595758 containing the EST sequence zc92h03.rl in the pBluescriptSK-vector was purchased from ATCC (Rockville, Md.). The DNA insertedinto the pBluescript SK-vector was further sequenced using T7 and T3vector promoters as well as sequence specific internal primers. The ESTencoded 440 amino acids showing a 24.4% identity to gp91phox, includinga stop codon corresponding to the C-terminus of gp91phox. 5′-RACE wascarried out using mRNA obtained from human colon carcinoma cells (CaCo2)and the Marathon cDNA Amplification Kit (ClonTech, Palo Alto). Thefollowing gene-specific primers were used for this procedure:5′-GAAGTGGTGGGAGGCGAAGACATA-3′ (SEQ ID NO:26) and5′-CCTGTCATACCTGGGACGGTCTGG-3′ (SEQ ID NO:27).

The results of the 5′-RACE yielded an additional 2 kilobase of sequencedDNA but this region did not contain the start codon. To complete thesequence of the 5′ and 3′ regions of duox2, 5′-RACE and 3′-RACE werecarried out using a human adult pancreas mRNA (Clontech, Palo Alto,Calif.) with the kit of 5′ RACE System for Rapid Amplification of cDNAEnds version 2.0 (Gibco BRL, Gaithersburg, Md.). PCR done using thefollowing specific primers resulted in a total predicted amino acidsequence of about 1000 residues: 5′-RACE: Primer 3:5′-GAGCACAGTGAGATGCCTGTTCAG-3′ (SEQ ID NO:28); Primer 4:5′-GGAAGGCAGCAGAGAGCAATGATG-3′ (SEQ ID NO:29) (for nested PCR); 3′-RACEPrimer 5: 5′-ACATCTGCGAGCGGCACTTCCAGA-3′ (SEQ ID NO:30) Primer 6:5′-AGCTCGTCAACAGGCAGGACCGAGC-3′ (SEQ ID NO:31) (for nested PCR).

EXAMPLE 19 Sequence Analysis and Cloning of the Human Duox1 cDNA (SEQ IDNO:45) Encoding for Production of the Human Duox1 Protein (SEQ ID NO:46)

A cDNA clone of duox1 was obtained as follows. A homologous 357-baseportion of an expressed sequence tag (EST nr80d12.s1; Genbank accessionno. AA641653) from an invasive human prostate was identified by usingthe partial duox2 predicted amino-acid sequence described above as aquery in a Blast search. The bacterial strain #1441736 containing theEST sequence nr80d12.s1 in the pBluescript SK-vector was purchased fromATCC (Rockville, Md.). The DNA inserted into the pBluescript SK-vectorwas further sequenced using T7 and T3 vector promoters as well assequence specific internal primers. The EST insert encoded 673 aminoacids with no start or stop codons present. Northern Blot analysis ofduox1 indicated the gene was about 5.5 kilobase pairs. To complete thesequence of 5′ and 3′ regions of duox1, 5′ RACE and 3′-RACE were carriedout using a human adult lung MRNA (Clontech, Palo Alto, Calif.) with thekit of 5′ RACE System for Rapid Amplification of cDNA Ends version 2.0(Gibco BRL, Gaithersburg, Md.). The RACE procedure was carried out usingthe following specific primers: 5′-RACE: Primer 5:5′-GCAGTGCATCCACATCTTCAGCAC-3′ (SEQ ID NO:32); Primer 6:5′-GAGAGCTCTGGAGACACTTGAGTTC-3′ (SEQ ID NO:33) (for nested PCR); 3′-RACEPrimer 7: 5′-CATGTTCTCTCTGGCTGACAAG-3′ (SEQ ID NO:34); Primer 8:5′-CACAATAGCGAGCTCCGCTTCACGC-3′ (SEQ ID NO:35) (for nested PCR). RACEprocedures were successful in completing the 5′ sequence and the 3′sequence of duox1. The open reading frame is approximately 4563 basepairs.

EXAMPLE 20 Tissue Expression of Duox1 and Duox2

Based on the duox1 sequence data, several specific primers were designed(Primer 1a: 5′-GCAGGACATCAACCCTGCACTCTC-3′ (SEQ ID NO:36); Primer 2a:5′-AATGACACTGTACTGGAGGCCACAG-3′ (SEQ ID NO:57); Primer 3a:5′-CTGCCATCTACCACACGGATCTGC-3′ (SEQ ID NO:58); Primer 4a:5′-CTTGCCATTCCAAAGCTTCCATGC-3′ (SEQ ID NO:59) and used these todetermine the tissue expression patterns of duox1 using Human MultipleTissue PCR Panels (Clontech, Palo Alto, Calif.). It was determined thatduox1 is expressed primarily in lung, testis, placenta, prostate,pancreas, fetal heart, fetal kidney, fetal liver, fetal lung, fetalskeletal muscle and thymus, with highest expression in adult and fetallung. Among 16 adult tissues and 8 fetal tissues tested, duox1expression in brain, heart, kidney, colon, ovary, thymus, fetal brainand fetal spleen appeared to be low.

Two duox2 specific primers were also used to determine the tissueexpression patterns of duox2 using Human Multiple Tissue PCR (polymerasechain reaction) Panels (Clontech, Palo Alto, Calif.). (Primer 1b:5′-GTACAAGTCAGGACAGTGGGTGCG-3′ (SEQ ID NO:60); Primer 2b:5′-TGGATGATGTCAGCCAGCCACTCA-3′ (SEQ ID NO:61)). Duox2 is expressedprimarily in lung, pancreas, placenta, colon, prostate, testis and fetaltissues, with highest expression in adult lung and fetal tissues. Among16 adult tissues and 8 fetal tissues tested, duox2 expression in brain,heart, kidney, liver, skeletal muscle, thymus and fetal brain appearedto be low.

EXAMPLE 21 Role of Duox1 and Duox2 in Collagen Crosslinking

To investigate a possible role for the human duox1 and duox2, the modelorganism Caenorhabditis elegans and a new reverse genetic tool, RNAinterference (RNAi), were used to “knock out” the homologues of duox inthis organism (Fire et al. (1998) Nature 391, 806-811). This techniqueinvolved injection of double stranded RNA encoding a segment of Ce-duox1or Ce-duox2 into gonads of C. elegans N2 hermaphrodites. Injected wormswere then allowed to lay eggs, and the harvested eggs were allowed todevelop and the F1 progeny were scored for phenotypes. This procedurehas been documented to “knock-out” the expression of the gene ofinterest (Fire et al. (1998) Nature 391, 806-811).

In the case of Ce-duox1 and Ce-duox2, the knockout animals resulted in acomplex phenotype including worms with large superficial blisters, shortor “dumpy” worms, worms with locomotion disorders, and worms withretained eggs and/or larvae. Because of the high identity betweenCe-duox1 and Ce-duox2, three different RNA constructs were predicted toknock out either both genes or Ce-duox2 alone. In all cases, essentiallythe same group of phenotypes was obtained. Most or all of thesephenotypes had been described previously in C. elegans mutated in thecollagen biosynthetic pathway. C. elegans has an extracellular structureknown as the cuticle, a complex sheath composed largely of cross-linkedcollagen, which functions as the exoskeleton of the nematode.Cross-linking of collagen in nematodes occurs in part by cross-linkingtyrosine residues, and peroxidases such as sea urchin ovoperoxidase andhuman myeloperoxidase have previously been shown to be capable ofcarrying out this reaction.

Based upon the similarities of the phenotypes obtained, the Ce-duox1/2knockout worms were examined for the presence of dityrosine linkages,using an HPLC methodology (Andersen, S. O. (1966) Acta Physiol. Scand.66, Suppl. 263-265; Abdelrahim et al. (1997) J. Chromatogr. B Biomed.Sci. Appl. 696, 175-182). It was determined that dityrosine linkages,while easily detected in the wild type worms, were almost completelylacking in the knockout worms. Thus, an inability to catalyze dityrosinecross-linking accounts for the phenotype of C. elegans failing toexpress Ce-duox1/2. These data support the concept that the duox enzymesin higher organisms can probably function in a similar manner tomodulate the extracellular milieu, possibly the extracellular matrixand/or the basement membrane.

All patents, publications and abstracts cited above are incorporatedherein by reference in their entirety. It should be understood that theforegoing relates only to preferred embodiments of the present inventionand that numerous modifications or alterations may be made thereinwithout departing from the spirit and the scope of the present inventionas defined in the following claims.

61 1 2609 DNA Homo sapiens modified_base (2025) n at position 2025 = anynucleotide 1 gctgatagca cagttctgtc cagagaagga aggcggaata aacttattcattcccaggaa 60 ctcttggggt aggtgtgtgt ttttcacatc ttaaaggctc acagaccctgcgctggacaa 120 atgttccatt cctgaaggac ctctccagaa tccggattgc tgaatcttccctgttgccta 180 gaagggctcc aaaccacctc ttgaca atg gga aac tgg gtg gtt aaccac tgg 233 Met Gly Asn Trp Val Val Asn His Trp 1 5 ttt tca gtt ttg tttctg gtt gtt tgg tta ggg ctg aat gtt ttc ctg 281 Phe Ser Val Leu Phe LeuVal Val Trp Leu Gly Leu Asn Val Phe Leu 10 15 20 25 ttt gtg gat gcc ttcctg aaa tat gag aag gcc gac aaa tac tac tac 329 Phe Val Asp Ala Phe LeuLys Tyr Glu Lys Ala Asp Lys Tyr Tyr Tyr 30 35 40 aca aga aaa atc ctt gggtca aca ttg gcc tgt gcc cga gcg tct gct 377 Thr Arg Lys Ile Leu Gly SerThr Leu Ala Cys Ala Arg Ala Ser Ala 45 50 55 ctc tgc ttg aat ttt aac agcacg ctg atc ctg ctt cct gtg tgt cgc 425 Leu Cys Leu Asn Phe Asn Ser ThrLeu Ile Leu Leu Pro Val Cys Arg 60 65 70 aat ctg ctg tcc ttc ctg agg ggcacc tgc tca ttt tgc agc cgc aca 473 Asn Leu Leu Ser Phe Leu Arg Gly ThrCys Ser Phe Cys Ser Arg Thr 75 80 85 ctg aga aag caa ttg gat cac aac ctcacc ttc cac aag ctg gtg gcc 521 Leu Arg Lys Gln Leu Asp His Asn Leu ThrPhe His Lys Leu Val Ala 90 95 100 105 tat atg atc tgc cta cat aca gctatt cac atc att gca cac ctg ttt 569 Tyr Met Ile Cys Leu His Thr Ala IleHis Ile Ile Ala His Leu Phe 110 115 120 aac ttt gac tgc tat agc aga agccga cag gcc aca gat ggc tcc ctt 617 Asn Phe Asp Cys Tyr Ser Arg Ser ArgGln Ala Thr Asp Gly Ser Leu 125 130 135 gcc tcc att ctc tcc agc cta tctcat gat gag aaa aag ggg ggt tct 665 Ala Ser Ile Leu Ser Ser Leu Ser HisAsp Glu Lys Lys Gly Gly Ser 140 145 150 tgg cta aat ccc atc cag tcc cgaaac acg aca gtg gag tat gtg aca 713 Trp Leu Asn Pro Ile Gln Ser Arg AsnThr Thr Val Glu Tyr Val Thr 155 160 165 ttc acc agc gtt gct ggt ctc actgga gtg atc atg aca ata gcc ttg 761 Phe Thr Ser Val Ala Gly Leu Thr GlyVal Ile Met Thr Ile Ala Leu 170 175 180 185 att ctc atg gta act tca gctact gag ttc atc cgg agg agt tat ttt 809 Ile Leu Met Val Thr Ser Ala ThrGlu Phe Ile Arg Arg Ser Tyr Phe 190 195 200 gaa gtc ttc tgg tat act caccac ctt ttt atc ttc tat atc ctt ggc 857 Glu Val Phe Trp Tyr Thr His HisLeu Phe Ile Phe Tyr Ile Leu Gly 205 210 215 tta ggg att cac ggc att ggtgga att gtc cgg ggt caa aca gag gag 905 Leu Gly Ile His Gly Ile Gly GlyIle Val Arg Gly Gln Thr Glu Glu 220 225 230 agc atg aat gag agt cat cctcgc aag tgt gca gag tct ttt gag atg 953 Ser Met Asn Glu Ser His Pro ArgLys Cys Ala Glu Ser Phe Glu Met 235 240 245 tgg gat gat cgt gac tcc cactgt agg cgc cct aag ttt gaa ggg cat 1001 Trp Asp Asp Arg Asp Ser His CysArg Arg Pro Lys Phe Glu Gly His 250 255 260 265 ccc cct gag tct tgg aagtgg atc ctt gca ccg gtc att ctt tat atc 1049 Pro Pro Glu Ser Trp Lys TrpIle Leu Ala Pro Val Ile Leu Tyr Ile 270 275 280 tgt gaa agg atc ctc cggttt tac cgc tcc cag cag aag gtt gtg att 1097 Cys Glu Arg Ile Leu Arg PheTyr Arg Ser Gln Gln Lys Val Val Ile 285 290 295 acc aag gtt gtt atg caccca tcc aaa gtt ttg gaa ttg cag atg aac 1145 Thr Lys Val Val Met His ProSer Lys Val Leu Glu Leu Gln Met Asn 300 305 310 aag cgt ggc ttc agc atggaa gtg ggg cag tat atc ttt gtt aat tgc 1193 Lys Arg Gly Phe Ser Met GluVal Gly Gln Tyr Ile Phe Val Asn Cys 315 320 325 ccc tca atc tct ctc ctggaa tgg cat cct ttt act ttg acc tct gct 1241 Pro Ser Ile Ser Leu Leu GluTrp His Pro Phe Thr Leu Thr Ser Ala 330 335 340 345 cca gag gaa gat ttcttc tcc att cat atc cga gca gca ggg gac tgg 1289 Pro Glu Glu Asp Phe PheSer Ile His Ile Arg Ala Ala Gly Asp Trp 350 355 360 aca gaa aat ctc ataagg gct ttc gaa caa caa tat tca cca att ccc 1337 Thr Glu Asn Leu Ile ArgAla Phe Glu Gln Gln Tyr Ser Pro Ile Pro 365 370 375 agg att gaa gtg gatggt ccc ttt ggc aca gcc agt gag gat gtt ttc 1385 Arg Ile Glu Val Asp GlyPro Phe Gly Thr Ala Ser Glu Asp Val Phe 380 385 390 cag tat gaa gtg gctgtg ctg gtt gga gca gga att ggg gtc acc ccc 1433 Gln Tyr Glu Val Ala ValLeu Val Gly Ala Gly Ile Gly Val Thr Pro 395 400 405 ttt gct tct atc ttgaaa tcc atc tgg tac aaa ttc cag tgt gca gac 1481 Phe Ala Ser Ile Leu LysSer Ile Trp Tyr Lys Phe Gln Cys Ala Asp 410 415 420 425 cac aac ctc aaaaca aaa aag atc tat ttc tac tgg atc tgc agg gag 1529 His Asn Leu Lys ThrLys Lys Ile Tyr Phe Tyr Trp Ile Cys Arg Glu 430 435 440 aca ggt gcc ttttcc tgg ttc aac aac ctg ttg act tcc ctg gaa cag 1577 Thr Gly Ala Phe SerTrp Phe Asn Asn Leu Leu Thr Ser Leu Glu Gln 445 450 455 gag atg gag gaatta ggc aaa gtg ggt ttt cta aac tac cgt ctc ttc 1625 Glu Met Glu Glu LeuGly Lys Val Gly Phe Leu Asn Tyr Arg Leu Phe 460 465 470 ctc acc gga tgggac agc aat att gtt ggt cat gca gca tta aac ttt 1673 Leu Thr Gly Trp AspSer Asn Ile Val Gly His Ala Ala Leu Asn Phe 475 480 485 gac aag gcc actgac atc gtg aca ggt ctg aaa cag aaa acc tcc ttt 1721 Asp Lys Ala Thr AspIle Val Thr Gly Leu Lys Gln Lys Thr Ser Phe 490 495 500 505 ggg aga ccaatg tgg gac aat gag ttt tct aca ata gct acc tcc cac 1769 Gly Arg Pro MetTrp Asp Asn Glu Phe Ser Thr Ile Ala Thr Ser His 510 515 520 ccc aag tctgta gtg gga gtt ttc tta tgt ggc cct cgg act ttg gca 1817 Pro Lys Ser ValVal Gly Val Phe Leu Cys Gly Pro Arg Thr Leu Ala 525 530 535 aag agc ctgcgc aaa tgc tgt cac cga tat tcc agt ctg gat cct aga 1865 Lys Ser Leu ArgLys Cys Cys His Arg Tyr Ser Ser Leu Asp Pro Arg 540 545 550 aag gtt caattc tac ttc aac aaa gaa aat ttt tga gttataggaa 1911 Lys Val Gln Phe TyrPhe Asn Lys Glu Asn Phe 555 560 565 taaggacggt aatctgcatt ttgtctctttgtatcttcag taattgagtt ataggaataa 1971 ggacggtaat ctgcattttg tctctttgtatcttcagtaa tttacttggt ctcntcaggt 2031 ttgancagtc actttaggat aagaatgtgcctctcaagcc ttgactccct ggtattcttt 2091 ttttgattgc attcaacttc gttacttgagcttcagcaac ttaagaactt ctgaagttct 2151 taaagttctg aanttcttaa agcccatggatcctttctca gaaaaataac tgtaaatctt 2211 tctggacagc catgactgta gcaaggcttgatagcagaag tttggtggtt canaattata 2271 caactaatcc caggtgattt tatcaattccagtgttacca tctcctgagt tttggtttgt 2331 aatcttttgt ccctcccacc cccacagaagattttaagta gggtgacttt ttaaataaaa 2391 atttattgaa taattaatga taaaacataataataaacat aaataataaa caaaattacc 2451 gagaacccca tccccatata acaccaacagtgtacatgtt tactgtcact tttgatatgg 2511 tttatccagt gtgaacagca atttattatttttgctcatc aaaaaataaa ggattttttt 2571 tcacttgaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaa 2609 2 564 PRT Homo sapiens 2 Met Gly Asn Trp Val Val Asn HisTrp Phe Ser Val Leu Phe Leu Val 1 5 10 15 Val Trp Leu Gly Leu Asn ValPhe Leu Phe Val Asp Ala Phe Leu Lys 20 25 30 Tyr Glu Lys Ala Asp Lys TyrTyr Tyr Thr Arg Lys Ile Leu Gly Ser 35 40 45 Thr Leu Ala Cys Ala Arg AlaSer Ala Leu Cys Leu Asn Phe Asn Ser 50 55 60 Thr Leu Ile Leu Leu Pro ValCys Arg Asn Leu Leu Ser Phe Leu Arg 65 70 75 80 Gly Thr Cys Ser Phe CysSer Arg Thr Leu Arg Lys Gln Leu Asp His 85 90 95 Asn Leu Thr Phe His LysLeu Val Ala Tyr Met Ile Cys Leu His Thr 100 105 110 Ala Ile His Ile IleAla His Leu Phe Asn Phe Asp Cys Tyr Ser Arg 115 120 125 Ser Arg Gln AlaThr Asp Gly Ser Leu Ala Ser Ile Leu Ser Ser Leu 130 135 140 Ser His AspGlu Lys Lys Gly Gly Ser Trp Leu Asn Pro Ile Gln Ser 145 150 155 160 ArgAsn Thr Thr Val Glu Tyr Val Thr Phe Thr Ser Val Ala Gly Leu 165 170 175Thr Gly Val Ile Met Thr Ile Ala Leu Ile Leu Met Val Thr Ser Ala 180 185190 Thr Glu Phe Ile Arg Arg Ser Tyr Phe Glu Val Phe Trp Tyr Thr His 195200 205 His Leu Phe Ile Phe Tyr Ile Leu Gly Leu Gly Ile His Gly Ile Gly210 215 220 Gly Ile Val Arg Gly Gln Thr Glu Glu Ser Met Asn Glu Ser HisPro 225 230 235 240 Arg Lys Cys Ala Glu Ser Phe Glu Met Trp Asp Asp ArgAsp Ser His 245 250 255 Cys Arg Arg Pro Lys Phe Glu Gly His Pro Pro GluSer Trp Lys Trp 260 265 270 Ile Leu Ala Pro Val Ile Leu Tyr Ile Cys GluArg Ile Leu Arg Phe 275 280 285 Tyr Arg Ser Gln Gln Lys Val Val Ile ThrLys Val Val Met His Pro 290 295 300 Ser Lys Val Leu Glu Leu Gln Met AsnLys Arg Gly Phe Ser Met Glu 305 310 315 320 Val Gly Gln Tyr Ile Phe ValAsn Cys Pro Ser Ile Ser Leu Leu Glu 325 330 335 Trp His Pro Phe Thr LeuThr Ser Ala Pro Glu Glu Asp Phe Phe Ser 340 345 350 Ile His Ile Arg AlaAla Gly Asp Trp Thr Glu Asn Leu Ile Arg Ala 355 360 365 Phe Glu Gln GlnTyr Ser Pro Ile Pro Arg Ile Glu Val Asp Gly Pro 370 375 380 Phe Gly ThrAla Ser Glu Asp Val Phe Gln Tyr Glu Val Ala Val Leu 385 390 395 400 ValGly Ala Gly Ile Gly Val Thr Pro Phe Ala Ser Ile Leu Lys Ser 405 410 415Ile Trp Tyr Lys Phe Gln Cys Ala Asp His Asn Leu Lys Thr Lys Lys 420 425430 Ile Tyr Phe Tyr Trp Ile Cys Arg Glu Thr Gly Ala Phe Ser Trp Phe 435440 445 Asn Asn Leu Leu Thr Ser Leu Glu Gln Glu Met Glu Glu Leu Gly Lys450 455 460 Val Gly Phe Leu Asn Tyr Arg Leu Phe Leu Thr Gly Trp Asp SerAsn 465 470 475 480 Ile Val Gly His Ala Ala Leu Asn Phe Asp Lys Ala ThrAsp Ile Val 485 490 495 Thr Gly Leu Lys Gln Lys Thr Ser Phe Gly Arg ProMet Trp Asp Asn 500 505 510 Glu Phe Ser Thr Ile Ala Thr Ser His Pro LysSer Val Val Gly Val 515 520 525 Phe Leu Cys Gly Pro Arg Thr Leu Ala LysSer Leu Arg Lys Cys Cys 530 535 540 His Arg Tyr Ser Ser Leu Asp Pro ArgLys Val Gln Phe Tyr Phe Asn 545 550 555 560 Lys Glu Asn Phe 3 2044 DNAHomo sapiens CDS (104)..(1810) 3 caaagacaaa ataatttact agggaagcccttactaacga cccaacatcc agacacaggt 60 gagggagaag aaatttcctg acagccgaagagcaacaagt atc atg atg ggg tgc 115 Met Met Gly Cys 1 tgg att ttg aat gagggt ctc tcc acc ata tta gta ctc tca tgg ctg 163 Trp Ile Leu Asn Glu GlyLeu Ser Thr Ile Leu Val Leu Ser Trp Leu 5 10 15 20 gga ata aat ttt tatctg ttt att gac acg ttc tac tgg tat gaa gag 211 Gly Ile Asn Phe Tyr LeuPhe Ile Asp Thr Phe Tyr Trp Tyr Glu Glu 25 30 35 gag gag tct ttc cat tacaca cga gtt att ttg ggt tca aca ctg gct 259 Glu Glu Ser Phe His Tyr ThrArg Val Ile Leu Gly Ser Thr Leu Ala 40 45 50 tgg gca cga gca tcc gca ctgtgc ctg aat ttt aac tgc atg cta att 307 Trp Ala Arg Ala Ser Ala Leu CysLeu Asn Phe Asn Cys Met Leu Ile 55 60 65 cta ata cct gtc agt cga aac cttatt tca ttc ata aga gga aca agt 355 Leu Ile Pro Val Ser Arg Asn Leu IleSer Phe Ile Arg Gly Thr Ser 70 75 80 att tgc tgc aga gga ccg tgg agg aggcaa tta gac aaa aac ctc aga 403 Ile Cys Cys Arg Gly Pro Trp Arg Arg GlnLeu Asp Lys Asn Leu Arg 85 90 95 100 ttt cac aaa ctg gtc gcc tat ggg atagct gtt aat gca acc atc cac 451 Phe His Lys Leu Val Ala Tyr Gly Ile AlaVal Asn Ala Thr Ile His 105 110 115 atc gtg gcg cat ttc ttc aac ctg gaacgc tac cac tgg agc cag tcc 499 Ile Val Ala His Phe Phe Asn Leu Glu ArgTyr His Trp Ser Gln Ser 120 125 130 gag gag gcc cag gga ctt ctg gcc gcactt tcc aag ctg ggc aac acc 547 Glu Glu Ala Gln Gly Leu Leu Ala Ala LeuSer Lys Leu Gly Asn Thr 135 140 145 cct aac gag agc tac ctc aac cct gtccgg acc ttc ccc aca aac aca 595 Pro Asn Glu Ser Tyr Leu Asn Pro Val ArgThr Phe Pro Thr Asn Thr 150 155 160 acc act gaa ttg cta agg aca ata gcaggc gtc acc ggt ctg gtg atc 643 Thr Thr Glu Leu Leu Arg Thr Ile Ala GlyVal Thr Gly Leu Val Ile 165 170 175 180 tct ctg gct tta gtc ttg atc atgacc tcg tca act gag ttc atc aga 691 Ser Leu Ala Leu Val Leu Ile Met ThrSer Ser Thr Glu Phe Ile Arg 185 190 195 cag gcc tcc tat gag ttg ttc tggtac aca cac cat gtt ttc atc gtc 739 Gln Ala Ser Tyr Glu Leu Phe Trp TyrThr His His Val Phe Ile Val 200 205 210 ttc ttt ctc agc ctg gcc atc catggg acg ggt cgg att gtt cga ggc 787 Phe Phe Leu Ser Leu Ala Ile His GlyThr Gly Arg Ile Val Arg Gly 215 220 225 caa acc caa gac agt ctc tct ctgcac aac atc acc ttc tgt aga gac 835 Gln Thr Gln Asp Ser Leu Ser Leu HisAsn Ile Thr Phe Cys Arg Asp 230 235 240 cgc tat gca gaa tgg cag aca gtggcc caa tgc ccc gtg cct caa ttt 883 Arg Tyr Ala Glu Trp Gln Thr Val AlaGln Cys Pro Val Pro Gln Phe 245 250 255 260 tct ggc aag gaa ccc tcg gcttgg aaa tgg att tta ggc cct gtg gtc 931 Ser Gly Lys Glu Pro Ser Ala TrpLys Trp Ile Leu Gly Pro Val Val 265 270 275 ttg tat gca tgt gaa aga ataatt agg ttc tgg cga ttt caa caa gaa 979 Leu Tyr Ala Cys Glu Arg Ile IleArg Phe Trp Arg Phe Gln Gln Glu 280 285 290 gtt gtc att acc aag gtg gtaagc cac ccc tct gga gtc ctg gaa ctt 1027 Val Val Ile Thr Lys Val Val SerHis Pro Ser Gly Val Leu Glu Leu 295 300 305 cac atg aaa aag cgt ggc tttaaa atg gcg cca ggg cag tac atc ttg 1075 His Met Lys Lys Arg Gly Phe LysMet Ala Pro Gly Gln Tyr Ile Leu 310 315 320 gtg cag tgc cca gcc ata tcttcg ctg gag tgg cac ccc ttc acc ctt 1123 Val Gln Cys Pro Ala Ile Ser SerLeu Glu Trp His Pro Phe Thr Leu 325 330 335 340 acc tct gcc ccc cag gaagac ttt ttc agc gtg cac atc cgg gca gca 1171 Thr Ser Ala Pro Gln Glu AspPhe Phe Ser Val His Ile Arg Ala Ala 345 350 355 gga gac tgg aca gca gcgcta ctg gag gcc ttt ggg gca gag gga cag 1219 Gly Asp Trp Thr Ala Ala LeuLeu Glu Ala Phe Gly Ala Glu Gly Gln 360 365 370 gcc ctc cag gag ccc tggagc ctg cca agg ctg gca gtg gac ggg ccc 1267 Ala Leu Gln Glu Pro Trp SerLeu Pro Arg Leu Ala Val Asp Gly Pro 375 380 385 ttt gga act gcc ctg acagat gta ttt cac tac cca gtg tgt gtg tgc 1315 Phe Gly Thr Ala Leu Thr AspVal Phe His Tyr Pro Val Cys Val Cys 390 395 400 gtt gcc gcg ggg atc ggagtc act ccc ttc gct gct ctt ctg aaa tct 1363 Val Ala Ala Gly Ile Gly ValThr Pro Phe Ala Ala Leu Leu Lys Ser 405 410 415 420 ata tgg tac aaa tgcagt gag gca cag acc cca ctg aag ctg agc aag 1411 Ile Trp Tyr Lys Cys SerGlu Ala Gln Thr Pro Leu Lys Leu Ser Lys 425 430 435 gtg tat ttc tac tggatt tgc cgg gat gca aga gct ttt gag tgg ttt 1459 Val Tyr Phe Tyr Trp IleCys Arg Asp Ala Arg Ala Phe Glu Trp Phe 440 445 450 gct gat ctc tta ctctcc ctg gaa aca cgg atg agt gag cag ggg aaa 1507 Ala Asp Leu Leu Leu SerLeu Glu Thr Arg Met Ser Glu Gln Gly Lys 455 460 465 act cac ttt ctg agttat cat ata ttt ctt acc ggc tgg gat gaa aat 1555 Thr His Phe Leu Ser TyrHis Ile Phe Leu Thr Gly Trp Asp Glu Asn 470 475 480 cag gct ctt cac atagct tta cac tgg gac gaa aat act gac gtg att 1603 Gln Ala Leu His Ile AlaLeu His Trp Asp Glu Asn Thr Asp Val Ile 485 490 495 500 aca ggc tta aagcag aag acc ttc tat ggg agg ccc aac tgg aac aat 1651 Thr Gly Leu Lys GlnLys Thr Phe Tyr Gly Arg Pro Asn Trp Asn Asn 505 510 515 gag ttc aag cagatt gcc tac aat cac ccc agc agc agt att ggc gtg 1699 Glu Phe Lys Gln IleAla Tyr Asn His Pro Ser Ser Ser Ile Gly Val 520 525 530 ttc ttc tgt ggacct aaa gct ctc tcg agg aca ctt caa aag atg tgc 1747 Phe Phe Cys Gly ProLys Ala Leu Ser Arg Thr Leu Gln Lys Met Cys 535 540 545 cac ttg tat tcatca gct gac ccc aga ggt gtt cat ttc tat tac aac 1795 His Leu Tyr Ser SerAla Asp Pro Arg Gly Val His Phe Tyr Tyr Asn 550 555 560 aag gag agc ttctag actttggagg tcaagtccag gcattgtgtt ttcaatcaag 1850 Lys Glu Ser Phe 565ttattgattc caaagaactc caccaggaat tcctgtgacg gcctgttgat atgagctccc 1910agttgggaac tggtgaataa taattaacta ttgtgaacag tacactatac catacttcct 1970tagcttataa ataacatgtc atatacaaca gaacaaaaac atttactgaa attaaaatat 2030attatgtttc tcca 2044 4 568 PRT Homo sapiens 4 Met Met Gly Cys Trp IleLeu Asn Glu Gly Leu Ser Thr Ile Leu Val 1 5 10 15 Leu Ser Trp Leu GlyIle Asn Phe Tyr Leu Phe Ile Asp Thr Phe Tyr 20 25 30 Trp Tyr Glu Glu GluGlu Ser Phe His Tyr Thr Arg Val Ile Leu Gly 35 40 45 Ser Thr Leu Ala TrpAla Arg Ala Ser Ala Leu Cys Leu Asn Phe Asn 50 55 60 Cys Met Leu Ile LeuIle Pro Val Ser Arg Asn Leu Ile Ser Phe Ile 65 70 75 80 Arg Gly Thr SerIle Cys Cys Arg Gly Pro Trp Arg Arg Gln Leu Asp 85 90 95 Lys Asn Leu ArgPhe His Lys Leu Val Ala Tyr Gly Ile Ala Val Asn 100 105 110 Ala Thr IleHis Ile Val Ala His Phe Phe Asn Leu Glu Arg Tyr His 115 120 125 Trp SerGln Ser Glu Glu Ala Gln Gly Leu Leu Ala Ala Leu Ser Lys 130 135 140 LeuGly Asn Thr Pro Asn Glu Ser Tyr Leu Asn Pro Val Arg Thr Phe 145 150 155160 Pro Thr Asn Thr Thr Thr Glu Leu Leu Arg Thr Ile Ala Gly Val Thr 165170 175 Gly Leu Val Ile Ser Leu Ala Leu Val Leu Ile Met Thr Ser Ser Thr180 185 190 Glu Phe Ile Arg Gln Ala Ser Tyr Glu Leu Phe Trp Tyr Thr HisHis 195 200 205 Val Phe Ile Val Phe Phe Leu Ser Leu Ala Ile His Gly ThrGly Arg 210 215 220 Ile Val Arg Gly Gln Thr Gln Asp Ser Leu Ser Leu HisAsn Ile Thr 225 230 235 240 Phe Cys Arg Asp Arg Tyr Ala Glu Trp Gln ThrVal Ala Gln Cys Pro 245 250 255 Val Pro Gln Phe Ser Gly Lys Glu Pro SerAla Trp Lys Trp Ile Leu 260 265 270 Gly Pro Val Val Leu Tyr Ala Cys GluArg Ile Ile Arg Phe Trp Arg 275 280 285 Phe Gln Gln Glu Val Val Ile ThrLys Val Val Ser His Pro Ser Gly 290 295 300 Val Leu Glu Leu His Met LysLys Arg Gly Phe Lys Met Ala Pro Gly 305 310 315 320 Gln Tyr Ile Leu ValGln Cys Pro Ala Ile Ser Ser Leu Glu Trp His 325 330 335 Pro Phe Thr LeuThr Ser Ala Pro Gln Glu Asp Phe Phe Ser Val His 340 345 350 Ile Arg AlaAla Gly Asp Trp Thr Ala Ala Leu Leu Glu Ala Phe Gly 355 360 365 Ala GluGly Gln Ala Leu Gln Glu Pro Trp Ser Leu Pro Arg Leu Ala 370 375 380 ValAsp Gly Pro Phe Gly Thr Ala Leu Thr Asp Val Phe His Tyr Pro 385 390 395400 Val Cys Val Cys Val Ala Ala Gly Ile Gly Val Thr Pro Phe Ala Ala 405410 415 Leu Leu Lys Ser Ile Trp Tyr Lys Cys Ser Glu Ala Gln Thr Pro Leu420 425 430 Lys Leu Ser Lys Val Tyr Phe Tyr Trp Ile Cys Arg Asp Ala ArgAla 435 440 445 Phe Glu Trp Phe Ala Asp Leu Leu Leu Ser Leu Glu Thr ArgMet Ser 450 455 460 Glu Gln Gly Lys Thr His Phe Leu Ser Tyr His Ile PheLeu Thr Gly 465 470 475 480 Trp Asp Glu Asn Gln Ala Leu His Ile Ala LeuHis Trp Asp Glu Asn 485 490 495 Thr Asp Val Ile Thr Gly Leu Lys Gln LysThr Phe Tyr Gly Arg Pro 500 505 510 Asn Trp Asn Asn Glu Phe Lys Gln IleAla Tyr Asn His Pro Ser Ser 515 520 525 Ser Ile Gly Val Phe Phe Cys GlyPro Lys Ala Leu Ser Arg Thr Leu 530 535 540 Gln Lys Met Cys His Leu TyrSer Ser Ala Asp Pro Arg Gly Val His 545 550 555 560 Phe Tyr Tyr Asn LysGlu Ser Phe 565 5 21 DNA Artificial Sequence Description of ArtificialSequence primer 5 aacaagcgtg gcttcagcat g 21 6 18 DNA ArtificialSequence Description of Artificial Sequence primer 6 agcaatattg ttggtcat18 7 24 DNA Artificial Sequence Description of Artificial Sequenceprimer 7 gacttgacag aaaatctata aggg 24 8 20 DNA Artificial SequenceDescription of Artificial Sequence primer 8 ttgtaccaga tggatttcaa 20 921 DNA Artificial Sequence Description of Artificial Sequence primer 9caggtctgaa acagaaaacc t 21 10 27 DNA Artificial Sequence Description ofArtificial Sequence primer 10 atgaattctc attaattatt caataaa 27 11 20 DNAArtificial Sequence Description of Artificial Sequence primer 11atctcaaaag actctgcaca 20 12 569 PRT Homo sapiens 12 Gly Asn Trp Ala ValAsn Glu Gly Leu Ser Ile Phe Ala Ile Leu Val 1 5 10 15 Trp Leu Gly LeuAsn Val Phe Leu Phe Val Trp Tyr Tyr Arg Val Tyr 20 25 30 Asp Ile Pro ProLys Phe Phe Tyr Thr Arg Lys Leu Leu Gly Ser Ala 35 40 45 Leu Ala Leu AlaArg Ala Pro Ala Ala Cys Leu Asn Phe Asn Cys Met 50 55 60 Leu Ile Leu LeuPro Val Cys Arg Asn Leu Leu Ser Phe Leu Arg Gly 65 70 75 80 Ser Ser AlaCys Cys Ser Thr Arg Val Arg Arg Gln Leu Asp Arg Asn 85 90 95 Leu Thr PheHis Lys Met Val Ala Trp Met Ile Ala Leu His Ser Ala 100 105 110 Ile HisThr Ile Ala His Leu Phe Asn Val Glu Trp Cys Val Asn Ala 115 120 125 ArgVal Asn Asn Ser Asp Pro Tyr Ser Val Ala Leu Ser Glu Leu Gly 130 135 140Asp Arg Gln Asn Glu Ser Tyr Leu Asn Phe Ala Arg Lys Arg Ile Lys 145 150155 160 Asn Pro Glu Gly Gly Leu Tyr Leu Ala Val Thr Leu Leu Ala Gly Ile165 170 175 Thr Gly Val Val Ile Thr Leu Cys Leu Ile Leu Ile Ile Thr SerSer 180 185 190 Thr Lys Thr Ile Arg Arg Ser Tyr Phe Glu Val Phe Trp TyrThr His 195 200 205 His Leu Phe Val Ile Phe Phe Ile Gly Leu Ala Ile HisGly Ala Glu 210 215 220 Arg Ile Val Arg Gly Gln Thr Ala Glu Ser Leu AlaVal His Asn Ile 225 230 235 240 Thr Val Cys Glu Gln Lys Ile Ser Glu TrpGly Lys Ile Lys Glu Cys 245 250 255 Pro Ile Pro Gln Phe Ala Gly Asn ProPro Met Thr Trp Lys Trp Ile 260 265 270 Val Gly Pro Met Phe Leu Tyr LeuCys Glu Arg Leu Val Arg Phe Trp 275 280 285 Arg Ser Gln Gln Lys Val ValIle Thr Lys Val Val Thr His Pro Phe 290 295 300 Lys Thr Ile Glu Leu GlnMet Lys Lys Lys Gly Phe Lys Met Glu Val 305 310 315 320 Gly Gln Tyr IlePhe Val Lys Cys Pro Lys Val Ser Lys Leu Glu Trp 325 330 335 His Pro PheThr Leu Thr Ser Ala Pro Glu Glu Asp Phe Phe Ser Ile 340 345 350 His IleArg Ile Val Gly Asp Trp Thr Glu Gly Leu Phe Asn Ala Cys 355 360 365 GlyCys Asp Lys Gln Glu Phe Gln Asp Ala Trp Lys Leu Pro Lys Ile 370 375 380Ala Val Asp Gly Pro Phe Gly Thr Ala Ser Glu Asp Val Phe Ser Tyr 385 390395 400 Glu Val Val Met Leu Val Gly Ala Gly Ile Gly Val Thr Pro Phe Ala405 410 415 Ser Ile Leu Lys Ser Val Trp Tyr Lys Tyr Cys Asn Asn Ala ThrAsn 420 425 430 Leu Lys Leu Lys Lys Ile Tyr Phe Tyr Trp Leu Cys Arg AspThr His 435 440 445 Ala Phe Glu Trp Phe Ala Asp Leu Leu Gln Leu Leu GluSer Gln Met 450 455 460 Gln Glu Arg Asn Asn Ala Gly Phe Leu Ser Tyr AsnIle Tyr Leu Thr 465 470 475 480 Gly Trp Asp Glu Ser Gln Ala Asn His PheAla Val His His Asp Glu 485 490 495 Glu Lys Asp Val Ile Thr Gly Leu LysGln Lys Thr Leu Tyr Gly Arg 500 505 510 Pro Asn Trp Asp Asn Glu Phe LysThr Ile Ala Ser Gln His Pro Asn 515 520 525 Thr Arg Ile Gly Val Phe LeuCys Gly Pro Glu Ala Leu Ala Glu Thr 530 535 540 Leu Ser Lys Gln Ser IleSer Asn Ser Glu Ser Gly Pro Arg Gly Val 545 550 555 560 His Phe Ile PheAsn Lys Glu Asn Phe 565 13 18 DNA Artificial Sequence Description ofArtificial Sequence primer 13 ttggctaaat cccatcca 18 14 21 DNAArtificial Sequence Description of Artificial Sequence primer 14tgcatgacca acaatattgc t 21 15 27 DNA Artificial Sequence Description ofArtificial Sequence primer 15 caaggtacct cttgaccatg ggaaact 27 16 27 DNAArtificial Sequence Description of Artificial Sequence primer 16acgaattcaa gtaaattact gaagata 27 17 26 DNA Artificial Sequencemodified_base (3) n at position 3 = inosine 17 ccngtntgtc gnaatctgctstcctt 26 18 29 DNA Artificial Sequence modified_base (5) n at position5 = inosine 18 tcccngcana nccagtagaa rtagatctt 29 19 26 DNA ArtificialSequence Description of Artificial Sequence primer 19 ttggcacagtcagtgaggat gtcttc 26 20 30 DNA Artificial Sequence Description ofArtificial Sequence primer 20 ctgttggctt ctactgtagc gttcaaagtt 30 21 563PRT Rat 21 Met Gly Asn Trp Leu Val Asn His Trp Leu Ser Val Leu Phe LeuVal 1 5 10 15 Ser Trp Leu Gly Leu Asn Ile Phe Leu Phe Val Tyr Val PheLeu Asn 20 25 30 Tyr Glu Lys Ser Asp Lys Tyr Tyr Tyr Thr Arg Glu Ile LeuGly Thr 35 40 45 Ala Leu Ala Leu Ala Arg Ala Ser Ala Leu Cys Leu Asn PheAsn Ser 50 55 60 Met Val Ile Leu Ile Pro Val Cys Arg Asn Leu Leu Ser PheLeu Arg 65 70 75 80 Gly Thr Cys Ser Phe Cys Asn His Thr Leu Arg Lys ProLeu Asp His 85 90 95 Asn Leu Thr Phe His Lys Leu Val Ala Tyr Met Ile CysIle Phe Thr 100 105 110 Ala Ile His Ile Ile Ala His Leu Phe Asn Phe GluArg Tyr Ser Arg 115 120 125 Ser Gln Gln Ala Met Asp Gly Ser Leu Ala SerVal Leu Ser Ser Leu 130 135 140 Phe His Pro Glu Lys Glu Asp Ser Trp LeuAsn Pro Ile Gln Ser Pro 145 150 155 160 Asn Val Thr Val Met Tyr Ala AlaPhe Thr Ser Ile Ala Gly Leu Thr 165 170 175 Gly Val Val Ala Thr Val AlaLeu Val Leu Met Val Thr Ser Ala Met 180 185 190 Glu Phe Ile Arg Arg AsnTyr Phe Glu Leu Phe Trp Tyr Thr His His 195 200 205 Leu Phe Ile Ile TyrIle Ile Cys Leu Gly Ile His Gly Leu Gly Gly 210 215 220 Ile Val Arg GlyGln Thr Glu Glu Ser Met Ser Glu Ser His Pro Arg 225 230 235 240 Asn CysSer Tyr Ser Phe His Glu Trp Asp Lys Tyr Glu Arg Ser Cys 245 250 255 ArgSer Pro His Phe Val Gly Gln Pro Pro Glu Ser Trp Lys Trp Ile 260 265 270Leu Ala Pro Ile Ala Phe Tyr Ile Phe Glu Arg Ile Leu Arg Phe Tyr 275 280285 Arg Ser Arg Gln Lys Val Val Ile Thr Lys Val Val Met His Pro Cys 290295 300 Lys Val Leu Glu Leu Gln Met Arg Lys Arg Gly Phe Thr Met Gly Ile305 310 315 320 Gly Gln Tyr Ile Phe Val Asn Cys Pro Ser Ile Ser Phe LeuGlu Trp 325 330 335 His Pro Phe Thr Leu Thr Ser Ala Pro Glu Glu Glu PhePhe Ser Ile 340 345 350 His Ile Arg Ala Ala Gly Asp Trp Thr Glu Asn LeuIle Arg Thr Phe 355 360 365 Glu Gln Gln His Ser Pro Met Pro Arg Ile GluVal Asp Gly Pro Phe 370 375 380 Gly Thr Val Ser Glu Asp Val Phe Gln TyrGlu Val Ala Val Leu Val 385 390 395 400 Gly Ala Gly Ile Gly Val Thr ProPhe Ala Ser Phe Leu Lys Ser Ile 405 410 415 Trp Tyr Lys Phe Gln Arg AlaHis Asn Lys Leu Lys Thr Gln Lys Ile 420 425 430 Tyr Phe Tyr Trp Ile CysArg Glu Thr Gly Ala Phe Ala Trp Phe Asn 435 440 445 Asn Leu Leu Asn SerLeu Glu Gln Glu Met Asp Glu Leu Gly Lys Pro 450 455 460 Asp Phe Leu AsnTyr Arg Leu Phe Leu Thr Gly Trp Asp Ser Asn Ile 465 470 475 480 Ala GlyHis Ala Ala Leu Asn Phe Asp Arg Ala Thr Asp Val Leu Thr 485 490 495 GlyLeu Lys Gln Lys Thr Ser Phe Gly Arg Pro Met Trp Asp Asn Glu 500 505 510Phe Ser Arg Ile Ala Thr Ala His Pro Lys Ser Val Val Gly Val Phe 515 520525 Leu Cys Gly Pro Pro Thr Leu Ala Lys Ser Leu Arg Lys Cys Cys Arg 530535 540 Arg Tyr Ser Ser Leu Asp Pro Arg Lys Val Gln Phe Tyr Phe Asn Lys545 550 555 560 Glu Thr Phe 22 2577 DNA Rat 22 ttctgagtag gtgtgcatttgagtgtcata aagacatata tcttgagcta gacagaagtt 60 cctatcctga aggatcccatcagagaaacc agattgctcc taagaggctc cagacctcca 120 tttgacaatg ggaaactggctggttaacca ctggctctca gttttgtttc tggtttcttg 180 gttggggctg aacatttttctgtttgtgta cgtcttcctg aattatgaga agtctgacaa 240 gtactattac acgagagaaattctcggaac tgccttggcc ttggccagag catctgcttt 300 gtgcctgaat tttaacagcatggtgatcct gattcctgtg tgtcgaaatc tgctctcctt 360 cctgaggggc acctgctcattttgcaacca cacgctgaga aagccattgg atcacaacct 420 caccttccat aagctggtggcatatatgat ctgcatattc acagctattc atatcattgc 480 acatctattt aactttgaacgctacagtag aagccaacag gccatggatg gatctcttgc 540 ctctgttctc tccagcctattccatcccga gaaagaagat tcttggctaa atcccatcca 600 gtctccaaac gtgacagtgatgtatgcagc atttaccagt attgctggcc ttactggagt 660 ggtcgccact gtggctttggttctcatggt aacttcagct atggagttta tccgcaggaa 720 ttattttgag ctcttctggtatacacatca ccttttcatc atctatatca tctgcttagg 780 gatccatggc ctgggggggattgtccgggg tcaaacagaa gagagcatga gtgaaagtca 840 tccccgcaac tgttcatactctttccacga gtgggataag tatgaaagga gttgcaggag 900 tcctcatttt gtggggcaaccccctgagtc ttggaagtgg atcctcgcgc cgattgcttt 960 ttatatcttt gaaaggatccttcgctttta tcgctcccgg cagaaggtcg tgattaccaa 1020 ggttgtcatg cacccatgtaaagttttgga attgcagatg aggaagcggg gctttactat 1080 gggaatagga cagtatatattcgtaaattg cccctcgatt tccttcctgg aatggcatcc 1140 ctttactctg acctctgctccagaggaaga atttttctcc attcatattc gagcagcagg 1200 ggactggaca gaaaatctcataaggacatt tgaacaacag cactcaccaa tgcccaggat 1260 cgaggtggat ggtccctttggcacagtcag tgaggatgtc ttccagtacg aagtggctgt 1320 actggttggg gcagggattggcgtcactcc ctttgcttcc ttcttgaaat ctatctggta 1380 caaattccag cgtgcacacaacaagctgaa aacacaaaag atctatttct actggatttg 1440 tagagagacg ggtgcctttgcctggttcaa caacttattg aattccctgg aacaagagat 1500 ggacgaatta ggcaaaccggatttcctaaa ctaccgactc ttcctcactg gctgggatag 1560 caacattgct ggtcatgcagcattaaactt tgacagagcc actgacgtcc tgacaggtct 1620 gaaacagaaa acctcctttgggagaccaat gtgggacaat gagttttcta gaatagctac 1680 tgcccacccc aagtctgtggtgggggtttt cttatgcggc cctccgactt tggcaaaaag 1740 cctgcgcaaa tgctgtcggcggtactcaag tctggatcct aggaaggttc aattctactt 1800 caacaaagaa acgttctgaattggaggaag ccgcacagta gtacttctcc atcttccttt 1860 tcactaacgt gtgggtcagctactagatag tccgttgtcg cacaaggact tcactcccat 1920 cttaaagttg actcaactccatcattcttg ggctttggca acatgagagc tgcataactc 1980 acaattgcaa aacacatgaattattattgg ggggattgta aatccttctg ggaaacctgc 2040 ctttagctga atcttgctggttgacacttg cacaatttaa cctcaggtgt cttggttgat 2100 acctgataat cttccctcccacctgtccct cacagaagat ttctaagtag ggtgatttta 2160 aaatatttat tgaatccacgacaaaacaat aatcataaat aataaacata aaattaccaa 2220 gattcccact cccatatcatacccactaag aacatcgtta tacatgagct tatcatccag 2280 tgtgaccaac aatttatactttactgtgcc aaaataatct tcatctttgc ttattgaaca 2340 attttgctga ctttccctagtaatatctta agtatattaa ctggaatcaa atttgtatta 2400 tagttagaag ccaactatattgccagtttg tattgtttga aataactgga aaggcctgac 2460 ctacatcgtg gggtaatttaacagaagctc tttccatttt ttgttgttgt tgttaaagag 2520 ttttgtttat gaatgtgttataaaaagaaa ataaaaagtt ataattttga cggaaaa 2577 23 332 PRT Homo sapiens 23Glu Ser Met Asn Glu Ser His Pro Arg Lys Cys Ala Glu Ser Phe Glu 1 5 1015 Met Trp Asp Asp Arg Asp Ser His Cys Arg Arg Pro Lys Phe Glu Gly 20 2530 His Pro Pro Glu Ser Trp Lys Trp Ile Leu Ala Pro Val Ile Leu Tyr 35 4045 Ile Cys Glu Arg Ile Leu Arg Phe Tyr Arg Ser Gln Gln Lys Val Val 50 5560 Ile Thr Lys Val Val Met His Pro Ser Lys Val Leu Glu Leu Gln Met 65 7075 80 Asn Lys Arg Gly Phe Ser Met Glu Val Gly Gln Tyr Ile Phe Val Asn 8590 95 Cys Pro Ser Ile Ser Leu Leu Glu Trp His Pro Phe Thr Leu Thr Ser100 105 110 Ala Pro Glu Glu Asp Phe Phe Ser Ile His Ile Arg Ala Ala GlyAsp 115 120 125 Trp Thr Glu Asn Leu Ile Arg Ala Phe Glu Gln Gln Tyr SerPro Ile 130 135 140 Pro Arg Ile Glu Val Asp Gly Pro Phe Gly Thr Ala SerGlu Asp Val 145 150 155 160 Phe Gln Tyr Glu Val Ala Val Leu Val Gly AlaGly Ile Gly Val Thr 165 170 175 Pro Phe Ala Ser Ile Leu Lys Ser Ile TrpTyr Lys Phe Gln Cys Ala 180 185 190 Asp His Asn Leu Lys Thr Lys Lys IleTyr Phe Tyr Trp Ile Cys Arg 195 200 205 Glu Thr Gly Ala Phe Ser Trp PheAsn Asn Leu Leu Thr Ser Leu Glu 210 215 220 Gln Glu Met Glu Glu Leu GlyLys Val Gly Phe Leu Asn Tyr Arg Leu 225 230 235 240 Phe Leu Thr Gly TrpAsp Ser Asn Ile Val Gly His Ala Ala Leu Asn 245 250 255 Phe Asp Lys AlaThr Asp Ile Val Thr Gly Leu Lys Gln Lys Thr Ser 260 265 270 Phe Gly ArgPro Met Trp Asp Asn Glu Phe Ser Thr Ile Ala Thr Ser 275 280 285 His ProLys Ser Val Val Gly Val Phe Leu Cys Gly Pro Arg Thr Leu 290 295 300 AlaLys Ser Leu Arg Lys Cys Cys His Arg Tyr Ser Ser Leu Asp Pro 305 310 315320 Arg Lys Val Gln Phe Tyr Phe Asn Lys Glu Asn Phe 325 330 24 14 PRTHomo sapiens 24 Cys Ala Glu Ser Phe Glu Met Trp Asp Asp Arg Asp Ser His1 5 10 25 14 PRT Homo sapiens 25 Lys Ser Leu Arg Lys Cys Cys His Arg TyrSer Ser Leu Asp 1 5 10 26 24 DNA Artificial Sequence Description ofArtificial Sequence primer 26 gaagtggtgg gaggcgaaga cata 24 27 24 DNAArtificial Sequence Description of Artificial Sequence primer 27cctgtcatac ctgggacggt ctgg 24 28 24 DNA Artificial Sequence Descriptionof Artificial Sequence primer 28 gagcacagtg agatgcctgt tcag 24 29 24 DNAArtificial Sequence Description of Artificial Sequence primer 29ggaaggcagc agagagcaat gatg 24 30 24 DNA Artificial Sequence Descriptionof Artificial Sequence primer 30 acatctgcga gcggcacttc caga 24 31 25 DNAArtificial Sequence Description of Artificial Sequence primer 31agctcgtcaa caggcaggac cgagc 25 32 24 DNA Artificial Sequence Descriptionof Artificial Sequence primer 32 gcagtgcatc cacatcttca gcac 24 33 25 DNAArtificial Sequence Description of Artificial Sequence primer 33gagagctctg gagacacttg agttc 25 34 22 DNA Artificial Sequence Descriptionof Artificial Sequence primer 34 catgttctct ctggctgaca ag 22 35 25 DNAArtificial Sequence Description of Artificial Sequence primer 35cacaatagcg agctccgctt cacgc 25 36 24 DNA Artificial Sequence Descriptionof Artificial Sequence primer 36 gcaggacatc aaccctgcac tctc 24 37 570PRT Bovine 37 Met Gly Asn Trp Val Val Asn Glu Gly Ile Ser Ile Phe ValIle Leu 1 5 10 15 Val Trp Leu Gly Met Asn Val Phe Leu Phe Val Trp TyrTyr Arg Val 20 25 30 Tyr Asp Ile Pro Asp Lys Phe Phe Tyr Thr Arg Lys LeuLeu Gly Ser 35 40 45 Ala Leu Ala Leu Ala Arg Ala Pro Ala Ala Cys Leu AsnPhe Asn Cys 50 55 60 Met Leu Ile Leu Leu Pro Val Cys Arg Asn Leu Leu SerPhe Leu Arg 65 70 75 80 Gly Ser Ser Ala Cys Cys Ser Thr Arg Ile Arg ArgGln Leu Asp Arg 85 90 95 Asn Leu Thr Phe His Lys Met Val Ala Trp Met IleAla Leu His Thr 100 105 110 Ala Ile His Thr Ile Ala His Leu Phe Asn ValGlu Trp Cys Val Asn 115 120 125 Ala Arg Val Asn Asn Ser Asp Pro Tyr SerIle Ala Leu Ser Asp Ile 130 135 140 Gly Asp Lys Pro Asn Glu Thr Tyr LeuAsn Phe Val Arg Gln Arg Ile 145 150 155 160 Lys Asn Pro Glu Gly Gly LeuTyr Val Ala Val Thr Arg Leu Ala Gly 165 170 175 Ile Thr Gly Val Val IleThr Leu Cys Leu Ile Leu Ile Ile Thr Ser 180 185 190 Ser Thr Lys Thr IleArg Arg Ser Tyr Phe Glu Val Phe Trp Tyr Thr 195 200 205 His His Leu PheVal Ile Phe Phe Ile Gly Leu Ala Ile His Gly Ala 210 215 220 Gln Arg IleVal Arg Gly Gln Thr Ala Glu Ser Leu Leu Lys His Gln 225 230 235 240 ProArg Asn Cys Tyr Gln Asn Ile Ser Gln Trp Gly Lys Ile Glu Asn 245 250 255Cys Pro Ile Pro Glu Phe Ser Gly Asn Pro Pro Met Thr Trp Lys Trp 260 265270 Ile Val Gly Pro Met Phe Leu Tyr Leu Cys Glu Arg Leu Val Arg Phe 275280 285 Trp Arg Ser Gln Gln Lys Val Val Ile Thr Lys Val Val Thr His Pro290 295 300 Phe Lys Thr Ile Glu Leu Gln Met Lys Lys Lys Gly Phe Lys MetGlu 305 310 315 320 Val Gly Gln Tyr Ile Phe Val Lys Cys Pro Val Val SerLys Leu Glu 325 330 335 Trp His Pro Phe Thr Leu Thr Ser Ala Pro Glu GluAsp Phe Phe Ser 340 345 350 Ile His Ile Arg Ile Val Gly Asp Trp Thr GluGly Leu Phe Lys Ala 355 360 365 Cys Gly Cys Asp Lys Gln Glu Phe Gln AspAla Trp Lys Leu Pro Lys 370 375 380 Ile Ala Val Asp Gly Pro Phe Gly ThrAla Ser Glu Asp Val Phe Ser 385 390 395 400 Tyr Glu Val Val Met Leu ValGly Ala Gly Ile Gly Val Thr Pro Phe 405 410 415 Ala Ser Ile Leu Lys SerVal Trp Tyr Lys Tyr Cys Asn Lys Ala Pro 420 425 430 Asn Leu Arg Leu LysLys Ile Tyr Phe Tyr Trp Leu Cys Arg Asp Thr 435 440 445 His Ala Phe GluTrp Phe Ala Asp Leu Leu Gln Leu Leu Glu Thr Gln 450 455 460 Met Gln GluLys Asn Asn Thr Asp Phe Leu Ser Tyr Asn Ile Cys Leu 465 470 475 480 ThrGly Trp Asp Glu Ser Gln Ala Ser His Phe Ala Met His His Asp 485 490 495Glu Glu Lys Asp Val Ile Thr Gly Leu Lys Gln Lys Thr Leu Tyr Gly 500 505510 Arg Pro Asn Trp Asp Asn Glu Phe Lys Thr Ile Gly Ser Gln His Pro 515520 525 Asn Thr Arg Ile Gly Val Phe Leu Cys Gly Pro Glu Ala Leu Ala Asp530 535 540 Thr Leu Asn Lys Gln Cys Ile Ser Asn Ser Asp Ser Gly Pro ArgGly 545 550 555 560 Val His Phe Ile Phe Asn Lys Glu Asn Phe 565 570 38570 PRT murine 38 Met Gly Asn Trp Ala Val Asn Glu Gly Leu Ser Ile PheVal Ile Leu 1 5 10 15 Val Trp Leu Gly Leu Asn Val Phe Leu Phe Ile AsnTyr Tyr Lys Val 20 25 30 Tyr Asp Asp Gly Pro Lys Tyr Asn Tyr Thr Arg LysLeu Leu Gly Ser 35 40 45 Ala Leu Ala Leu Ala Arg Ala Pro Ala Ala Cys LeuAsn Phe Asn Cys 50 55 60 Met Leu Ile Leu Leu Pro Val Cys Arg Asn Leu LeuSer Phe Leu Arg 65 70 75 80 Gly Ser Ser Ala Cys Cys Ser Thr Arg Ile ArgArg Gln Leu Asp Arg 85 90 95 Asn Leu Thr Phe His Lys Met Val Ala Trp MetIle Ala Leu His Thr 100 105 110 Ala Ile His Thr Ile Ala His Leu Phe AsnVal Glu Trp Cys Val Asn 115 120 125 Ala Arg Val Gly Ile Ser Asp Arg TyrSer Ile Ala Leu Ser Asp Ile 130 135 140 Gly Asp Asn Glu Asn Glu Glu TyrLeu Asn Phe Ala Arg Glu Lys Ile 145 150 155 160 Lys Asn Pro Glu Gly GlyLeu Tyr Val Ala Val Thr Arg Leu Ala Gly 165 170 175 Ile Thr Gly Ile ValIle Thr Leu Cys Leu Ile Leu Ile Ile Thr Ser 180 185 190 Ser Thr Lys ThrIle Arg Arg Ser Tyr Phe Glu Val Phe Trp Tyr Thr 195 200 205 His His LeuPhe Val Ile Phe Phe Ile Gly Leu Ala Ile His Gly Ala 210 215 220 Glu ArgIle Val Arg Gly Gln Thr Ala Glu Ser Leu Glu Glu His Asn 225 230 235 240Leu Asp Ile Cys Ala Asp Lys Ile Glu Glu Trp Gly Lys Ile Lys Glu 245 250255 Cys Pro Val Pro Lys Phe Ala Gly Asn Pro Pro Met Thr Trp Lys Trp 260265 270 Ile Val Gly Pro Met Phe Leu Tyr Leu Cys Glu Arg Leu Val Arg Phe275 280 285 Trp Arg Ser Gln Gln Lys Val Val Ile Thr Lys Val Val Thr HisPro 290 295 300 Phe Lys Thr Ile Glu Leu Gln Met Lys Lys Lys Gly Phe LysMet Glu 305 310 315 320 Val Gly Gln Tyr Ile Phe Val Lys Cys Pro Lys ValSer Lys Leu Glu 325 330 335 Trp His Pro Phe Thr Leu Thr Ser Ala Pro GluGlu Asp Phe Phe Ser 340 345 350 Ile His Ile Arg Ile Val Gly Asp Trp ThrGlu Gly Leu Phe Asn Ala 355 360 365 Cys Gly Cys Asp Lys Gln Glu Phe GlnAsp Ala Trp Lys Leu Pro Lys 370 375 380 Ile Ala Val Asp Gly Pro Phe GlyThr Ala Ser Glu Asp Val Phe Ser 385 390 395 400 Tyr Glu Val Val Met LeuVal Gly Ala Gly Ile Gly Val Thr Pro Phe 405 410 415 Ala Ser Ile Leu LysSer Val Trp Tyr Lys Tyr Cys Asp Asn Ala Thr 420 425 430 Ser Leu Lys LeuLys Lys Ile Tyr Phe Tyr Trp Leu Cys Arg Asp Thr 435 440 445 His Ala PheGlu Trp Phe Ala Asp Leu Leu Gln Leu Leu Glu Thr Gln 450 455 460 Met GlnGlu Arg Asn Asn Ala Asn Phe Leu Ser Tyr Asn Ile Tyr Leu 465 470 475 480Thr Gly Trp Asp Glu Ser Gln Ala Asn His Phe Ala Val His His Asp 485 490495 Glu Glu Lys Asp Val Ile Thr Gly Leu Lys Gln Lys Thr Leu Tyr Gly 500505 510 Arg Pro Asn Trp Asp Asn Glu Phe Lys Thr Ile Ala Ser Glu His Pro515 520 525 Asn Thr Thr Ile Gly Val Phe Leu Cys Gly Pro Glu Ala Leu AlaGlu 530 535 540 Thr Leu Ser Lys Gln Ser Ile Ser Asn Ser Glu Ser Gly ProArg Gly 545 550 555 560 Val His Phe Ile Phe Asn Lys Glu Asn Phe 565 57039 944 PRT Arabidopsis sp. 39 Met Lys Pro Phe Ser Lys Asn Asp Arg ArgArg Trp Ser Phe Asp Ser 1 5 10 15 Val Ser Ala Gly Lys Thr Ala Val GlySer Ala Ser Thr Ser Pro Gly 20 25 30 Thr Glu Tyr Ser Ile Asn Gly Asp GlnGlu Phe Val Glu Val Thr Ile 35 40 45 Asp Leu Gln Asp Asp Asp Thr Ile ValLeu Arg Ser Val Glu Pro Ala 50 55 60 Thr Ala Ile Asn Val Ile Gly Asp IleSer Asp Asp Asn Thr Gly Ile 65 70 75 80 Met Thr Pro Val Ser Ile Ser ArgSer Pro Thr Met Lys Arg Thr Ser 85 90 95 Ser Asn Arg Phe Arg Gln Phe SerGln Glu Leu Lys Ala Glu Ala Val 100 105 110 Ala Lys Ala Lys Gln Leu SerGln Glu Leu Lys Arg Phe Ser Trp Ser 115 120 125 Arg Ser Phe Ser Gly AsnLeu Thr Thr Thr Ser Thr Ala Ala Asn Gln 130 135 140 Ser Gly Gly Ala GlyGly Gly Leu Val Asn Ser Ala Leu Glu Ala Arg 145 150 155 160 Ala Leu ArgLys Gln Arg Ala Gln Leu Asp Arg Thr Arg Ser Ser Ala 165 170 175 Gln ArgAla Leu Arg Gly Leu Arg Phe Ile Ser Asn Lys Gln Lys Asn 180 185 190 ValAsp Gly Trp Asn Asp Val Gln Ser Asn Phe Glu Lys Phe Glu Lys 195 200 205Asn Gly Tyr Ile Tyr Arg Ser Asp Phe Ala Gln Cys Ile Gly Met Lys 210 215220 Asp Ser Lys Glu Phe Ala Leu Glu Leu Phe Asp Ala Leu Ser Arg Arg 225230 235 240 Arg Arg Leu Lys Val Glu Lys Ile Asn His Asp Glu Leu Tyr GluTyr 245 250 255 Trp Ser Gln Ile Asn Asp Glu Ser Phe Asp Ser Arg Leu GlnIle Phe 260 265 270 Phe Asp Ile Val Asp Lys Asn Glu Asp Gly Arg Ile ThrGlu Glu Glu 275 280 285 Val Lys Glu Ile Ile Met Leu Ser Ala Ser Ala AsnLys Leu Ser Arg 290 295 300 Leu Lys Glu Gln Ala Glu Glu Tyr Ala Ala LeuIle Met Glu Glu Leu 305 310 315 320 Asp Pro Glu Arg Leu Gly Tyr Ile GluLeu Trp Gln Leu Glu Thr Leu 325 330 335 Leu Leu Gln Lys Asp Thr Tyr LeuAsn Tyr Ser Gln Ala Leu Ser Tyr 340 345 350 Thr Ser Gln Ala Leu Ser GlnAsn Leu Gln Gly Leu Arg Gly Lys Ser 355 360 365 Arg Ile His Arg Met SerSer Asp Phe Val Tyr Ile Met Gln Glu Asn 370 375 380 Trp Lys Arg Ile TrpVal Leu Ser Leu Trp Ile Met Ile Met Ile Gly 385 390 395 400 Leu Phe LeuTrp Lys Phe Phe Gln Tyr Lys Gln Lys Asp Ala Phe His 405 410 415 Val MetGly Tyr Cys Leu Leu Thr Ala Lys Gly Ala Ala Glu Thr Leu 420 425 430 LysPhe Asn Met Ala Leu Ile Leu Phe Pro Val Cys Arg Asn Thr Ile 435 440 445Thr Trp Leu Arg Ser Thr Arg Leu Ser Tyr Phe Val Pro Phe Asp Asp 450 455460 Asn Ile Asn Phe His Lys Thr Ile Ala Gly Ala Ile Val Val Ala Val 465470 475 480 Ile Leu His Ile Gly Asp His Leu Ala Cys Asp Phe Pro Arg IleVal 485 490 495 Arg Ala Thr Glu Tyr Asp Tyr Asn Arg Tyr Leu Phe His TyrPhe Gln 500 505 510 Thr Lys Gln Pro Thr Tyr Phe Asp Leu Val Lys Gly ProGlu Gly Ile 515 520 525 Thr Gly Ile Leu Met Val Ile Leu Met Ile Ile SerPhe Thr Leu Ala 530 535 540 Thr Arg Trp Phe Arg Arg Asn Leu Val Lys LeuPro Lys Pro Phe Asp 545 550 555 560 Arg Leu Thr Gly Phe Asn Ala Phe TrpTyr Ser His His Leu Phe Val 565 570 575 Ile Val Tyr Ile Leu Leu Ile LeuHis Gly Ile Phe Leu Tyr Phe Ala 580 585 590 Lys Pro Trp Tyr Val Arg ThrThr Trp Met Tyr Leu Ala Val Pro Val 595 600 605 Leu Leu Tyr Gly Gly GluArg Thr Leu Arg Tyr Phe Arg Ser Gly Ser 610 615 620 Tyr Ser Val Arg LeuLeu Lys Val Ala Ile Tyr Pro Gly Asn Val Leu 625 630 635 640 Thr Leu GlnMet Ser Lys Pro Thr Gln Phe Arg Tyr Lys Ser Gly Gln 645 650 655 Tyr MetPhe Val Gln Cys Pro Ala Val Ser Pro Phe Glu Trp His Pro 660 665 670 PheSer Ile Thr Ser Ala Pro Glu Asp Asp Tyr Ile Ser Ile His Ile 675 680 685Arg Gln Leu Gly Asp Trp Thr Gln Glu Leu Lys Arg Val Phe Ser Glu 690 695700 Val Cys Glu Pro Pro Val Gly Gly Lys Ser Gly Leu Leu Arg Ala Asp 705710 715 720 Glu Thr Thr Lys Lys Ser Leu Pro Lys Leu Leu Ile Asp Gly ProTyr 725 730 735 Gly Ala Pro Ala Gln Asp Tyr Arg Lys Tyr Asp Val Leu LeuLeu Val 740 745 750 Gly Leu Gly Ile Gly Ala Thr Pro Phe Ile Ser Ile LeuLys Asp Leu 755 760 765 Leu Asn Asn Ile Val Lys Met Glu Glu His Ala AspSer Ile Ser Asp 770 775 780 Phe Ser Arg Ser Ser Glu Tyr Ser Thr Gly SerAsn Gly Asp Thr Pro 785 790 795 800 Arg Arg Lys Arg Ile Leu Lys Thr ThrAsn Ala Tyr Phe Tyr Trp Val 805 810 815 Thr Arg Glu Gln Gly Ser Phe AspTrp Phe Lys Gly Val Met Asn Glu 820 825 830 Val Ala Glu Leu Asp Gln ArgGly Val Ile Glu Met His Asn Tyr Leu 835 840 845 Thr Ser Val Tyr Glu GluGly Asp Ala Arg Ser Ala Leu Ile Thr Met 850 855 860 Val Gln Ala Leu AsnHis Ala Lys Asn Gly Val Asp Ile Val Ser Gly 865 870 875 880 Thr Arg ValArg Thr His Phe Ala Arg Pro Asn Trp Lys Lys Val Leu 885 890 895 Thr LysLeu Ser Ser Lys His Cys Asn Ala Arg Ile Gly Val Phe Tyr 900 905 910 CysGly Val Pro Val Leu Gly Lys Glu Leu Ser Lys Leu Cys Asn Thr 915 920 925Phe Asn Gln Lys Gly Ser Thr Lys Phe Glu Phe His Lys Glu His Phe 930 935940 40 590 PRT Rice 40 Asn Leu Ala Gly Leu Arg Lys Lys Ser Ser Ile ArgLys Ile Ser Thr 1 5 10 15 Ser Leu Ser Tyr Tyr Phe Glu Asp Asn Trp LysArg Leu Trp Val Leu 20 25 30 Ala Leu Trp Ile Gly Ile Met Ala Gly Leu PheThr Trp Lys Phe Met 35 40 45 Gln Tyr Arg Asn Arg Tyr Val Phe Asp Val MetGly Tyr Cys Val Thr 50 55 60 Thr Ala Lys Gly Ala Ala Glu Thr Leu Lys LeuAsn Met Ala Ile Ile 65 70 75 80 Leu Leu Pro Val Cys Arg Asn Thr Ile ThrTrp Leu Arg Ser Thr Arg 85 90 95 Ala Ala Arg Ala Leu Pro Phe Asp Asp AsnIle Asn Phe His Lys Thr 100 105 110 Ile Ala Ala Ala Ile Val Val Gly IleIle Leu His Ala Gly Asn His 115 120 125 Leu Val Cys Asp Phe Pro Arg LeuIle Lys Ser Ser Asp Glu Lys Tyr 130 135 140 Ala Pro Leu Gly Gln Tyr PheGly Glu Ile Lys Pro Thr Tyr Phe Thr 145 150 155 160 Leu Val Lys Gly ValGlu Gly Ile Thr Gly Val Ile Met Val Val Cys 165 170 175 Met Ile Ile AlaPhe Thr Leu Ala Thr Arg Trp Phe Arg Arg Ser Leu 180 185 190 Val Lys LeuPro Arg Pro Phe Asp Lys Leu Thr Gly Phe Asn Ala Phe 195 200 205 Trp TyrSer His His Leu Phe Ile Ile Val Tyr Ile Ala Leu Ile Val 210 215 220 HisGly Glu Cys Leu Tyr Leu Ile His Val Trp Tyr Arg Arg Thr Thr 225 230 235240 Trp Met Tyr Leu Ser Val Pro Val Cys Leu Tyr Val Gly Glu Arg Ile 245250 255 Leu Arg Phe Phe Arg Ser Gly Ser Tyr Ser Val Arg Leu Leu Lys Val260 265 270 Ala Ile Tyr Pro Gly Asn Val Leu Thr Leu Gln Met Ser Lys ProPro 275 280 285 Thr Phe Arg Tyr Lys Ser Gly Gln Tyr Met Phe Val Gln CysPro Ala 290 295 300 Val Ser Pro Phe Glu Trp His Pro Phe Ser Ile Thr SerAla Pro Gly 305 310 315 320 Asp Asp Tyr Leu Ser Ile His Val Arg Gln LeuGly Asp Trp Thr Arg 325 330 335 Glu Leu Lys Arg Val Phe Ala Ala Ala CysGlu Pro Pro Ala Gly Gly 340 345 350 Lys Ser Gly Leu Leu Arg Ala Asp GluThr Thr Lys Lys Ile Leu Pro 355 360 365 Lys Leu Leu Ile Asp Gly Pro TyrGly Ser Pro Ala Gln Asp Tyr Ser 370 375 380 Lys Tyr Asp Val Leu Leu LeuVal Gly Leu Gly Ile Gly Ala Thr Pro 385 390 395 400 Phe Ile Ser Ile LeuLys Asp Leu Leu Asn Asn Ile Ile Lys Met Glu 405 410 415 Glu Glu Glu AspAla Ser Thr Asp Leu Tyr Pro Pro Met Gly Arg Asn 420 425 430 Asn Pro HisVal Asp Leu Gly Thr Leu Met Thr Ile Thr Ser Arg Pro 435 440 445 Lys LysIle Leu Lys Thr Thr Asn Ala Tyr Phe Tyr Trp Val Thr Arg 450 455 460 GluGln Gly Ser Phe Asp Trp Phe Lys Gly Val Met Asn Glu Ile Ala 465 470 475480 Asp Leu Asp Gln Arg Asn Ile Ile Glu Met His Asn Tyr Leu Thr Ser 485490 495 Val Tyr Glu Glu Gly Asp Ala Arg Ser Ala Leu Ile Thr Met Leu Gln500 505 510 Ala Leu Asn His Ala Lys Asn Gly Val Asp Ile Val Ser Gly ThrLys 515 520 525 Val Arg Thr His Phe Ala Arg Pro Asn Trp Arg Lys Val LeuSer Lys 530 535 540 Ile Ser Ser Lys His Pro Tyr Ala Lys Ile Gly Val PheTyr Cys Gly 545 550 555 560 Ala Pro Val Leu Ala Gln Glu Leu Ser Lys LeuCys His Glu Phe Asn 565 570 575 Gly Lys Cys Thr Thr Lys Phe Asp Phe HisLys Glu His Phe 580 585 590 41 2619 DNA Rat 41 gtgctgtcag agctttacagagcctctggg catgcgcatg gctacccatt tcattgattt 60 acagaagtca tgctaaaatctctttcatgc atgtcttcct ttttcagtct ctcctttccc 120 aaagcttttc agtttgccctttgcttgtac caactgctat ccctcctcaa aggctgctgc 180 aaaaggtatg cctttttcttggaggctttc agcaaatact acctgggaac ctgcttcagc 240 tcttggaata tttaagtgaagagaacattt catagcattt gtatctttct ttgaaggagc 300 caccagacag actgccttggccttggccag agcatctgct ttgtgcctga attttaacag 360 catggtgatc ctgattcctgtgtgtcgaaa tctgctctcc ttcctgaggg gcacctgctc 420 attttgcaac cacacgctgagaaagccatt ggatcacaac ctcaccttcc ataagctggt 480 ggcatatatg atctgcatattcacagctat tcatatcatt gcacatctat ttaactttga 540 acgctacagt agaagccaacaggccatgga tggatctctt gcctctgttc tctccagcct 600 attccatccc gagaaagaagattcttggct aaatcccatc cagtctccaa acgtgacagt 660 gatgtatgca gcatttaccagtattgctgg ccttactgga gtggtcgcca ctgtggcttt 720 ggttctcatg gtaacttcagctatggagtt tatccgcagg aattattttg agctcttctg 780 gtatacacat caccttttcatcatctatat catctgctta gggatccatg gcctgggggg 840 gattgtccgg ggtcaaacagaagagagcat gagtgaaagt catccccgca actgttcata 900 ctctttccac gagtgggataagtatgaaag gagttgcagg agtcctcatt ttgtggggca 960 accccctgag tcttggaagtggatcctcgc gccgattgct ttttatatct ttgaaaggat 1020 ccttcgcttt tatcgctcccggcagaaggt cgtgattacc aaggttgtca tgcacccatg 1080 taaagttttg gaattgcagatgaggaagcg gggctttact atgggaatag gacagtatat 1140 attcgtaaat tgcccctcgatttccttcct ggaatggcat ccctttactc tgacctctgc 1200 tccagaggaa gaatttttctccattcatat tcgagcagca ggggactgga cagaaaatct 1260 cataaggaca tttgaacaacagcactcacc aatgcccagg atcgaggtgg atggtccctt 1320 tggcacagtc agtgaggatgtcttccagta cgaagtggct gtactggttg gggcagggat 1380 tggcgtcact ccctttgcttccttcttgaa atctatctgg tacaaattcc agcgtgcaca 1440 caacaagctg aaaacacaaaagatctattt ctactggatt tgtagagaga cgggtgcctt 1500 tgcctggttc aacaacttattgaattccct ggaacaagag atggacgaat taggcaaacc 1560 ggatttccta aactaccgactcttcctcac tggctgggat agcaacattg ctggtcatgc 1620 agcattaaac tttgacagagccactgacgt cctgacaggt ctgaaacaga aaacctcctt 1680 tgggagacca atgtgggacaatgagttttc tagaatagct actgcccacc ccaagtctgt 1740 ggtgggggtt ttcttatgcggccctccgac tttggcaaaa agcctgcgca aatgctgtcg 1800 gcggtactca agtctggatcctaggaaggt tcaattctac ttcaacaaag aaacgttctg 1860 aattggagga agccgcacagtagtacttct ccatcttcct tttcactaac gtgtgggtca 1920 gctactagat agtccgttgtcgcacaagga cttcactccc atcttaaagt tgactcaact 1980 ccatcattct tgggctttggcaacatgaga gctgcataac tcacaattgc aaaacacatg 2040 aattattatt ggggggattgtaaatccttc tgggaaacct gcctttagct gaatcttgct 2100 ggttgacact tgcacaatttaacctcaggt gtcttggttg atacctgata atcttccctc 2160 ccacctgtcc ctcacagaagatttctaagt agggtgattt taaaatattt attgaatcca 2220 cgacaaaaca ataatcataaataataaaca taaaattacc aagattccca ctcccatatc 2280 atacccacta agaacatcgttatacatgag cttatcatcc agtgtgacca acaatttata 2340 ctttactgtg ccaaaataatcttcatcttt gcttattgaa caattttgct gactttccct 2400 agtaatatct taagtatattaactggaatc aaatttgtat tatagttaga agccaactat 2460 attgccagtt tgtattgtttgaaataactg gaaaggcctg acctacatcg tggggtaatt 2520 taacagaagc tctttccattttttgttgtt gttgttaaag agttttgttt atgaatgtgt 2580 tataaaaaga aaataaaaagttataatttt gacggaaaa 2619 42 499 PRT Rat 42 Met Val Ile Leu Ile Pro ValCys Arg Asn Leu Leu Ser Phe Leu Arg 1 5 10 15 Gly Thr Cys Ser Phe CysAsn His Thr Leu Arg Lys Pro Leu Asp His 20 25 30 Asn Leu Thr Phe His LysLeu Val Ala Tyr Met Ile Cys Ile Phe Thr 35 40 45 Ala Ile His Ile Ile AlaHis Leu Phe Asn Phe Glu Arg Tyr Ser Arg 50 55 60 Ser Gln Gln Ala Met AspGly Ser Leu Ala Ser Val Leu Ser Ser Leu 65 70 75 80 Phe His Pro Glu LysGlu Asp Ser Trp Leu Asn Pro Ile Gln Ser Pro 85 90 95 Asn Val Thr Val MetTyr Ala Ala Phe Thr Ser Ile Ala Gly Leu Thr 100 105 110 Gly Val Val AlaThr Val Ala Leu Val Leu Met Val Thr Ser Ala Met 115 120 125 Glu Phe IleArg Arg Asn Tyr Phe Glu Leu Phe Trp Tyr Thr His His 130 135 140 Leu PheIle Ile Tyr Ile Ile Cys Leu Gly Ile His Gly Leu Gly Gly 145 150 155 160Ile Val Arg Gly Gln Thr Glu Glu Ser Met Ser Glu Ser His Pro Arg 165 170175 Asn Cys Ser Tyr Ser Phe His Glu Trp Asp Lys Tyr Glu Arg Ser Cys 180185 190 Arg Ser Pro His Phe Val Gly Gln Pro Pro Glu Ser Trp Lys Trp Ile195 200 205 Leu Ala Pro Ile Ala Phe Tyr Ile Phe Glu Arg Ile Leu Arg PheTyr 210 215 220 Arg Ser Arg Gln Lys Val Val Ile Thr Lys Val Val Met HisPro Cys 225 230 235 240 Lys Val Leu Glu Leu Gln Met Arg Lys Arg Gly PheThr Met Gly Ile 245 250 255 Gly Gln Tyr Ile Phe Val Asn Cys Pro Ser IleSer Phe Leu Glu Trp 260 265 270 His Pro Phe Thr Leu Thr Ser Ala Pro GluGlu Glu Phe Phe Ser Ile 275 280 285 His Ile Arg Ala Ala Gly Asp Trp ThrGlu Asn Leu Ile Arg Thr Phe 290 295 300 Glu Gln Gln His Ser Pro Met ProArg Ile Glu Val Asp Gly Pro Phe 305 310 315 320 Gly Thr Val Ser Glu AspVal Phe Gln Tyr Glu Val Ala Val Leu Val 325 330 335 Gly Ala Gly Ile GlyVal Thr Pro Phe Ala Ser Phe Leu Lys Ser Ile 340 345 350 Trp Tyr Lys PheGln Arg Ala His Asn Lys Leu Lys Thr Gln Lys Ile 355 360 365 Tyr Phe TyrTrp Ile Cys Arg Glu Thr Gly Ala Phe Ala Trp Phe Asn 370 375 380 Asn LeuLeu Asn Ser Leu Glu Gln Glu Met Asp Glu Leu Gly Lys Pro 385 390 395 400Asp Phe Leu Asn Tyr Arg Leu Phe Leu Thr Gly Trp Asp Ser Asn Ile 405 410415 Ala Gly His Ala Ala Leu Asn Phe Asp Arg Ala Thr Asp Val Leu Thr 420425 430 Gly Leu Lys Gln Lys Thr Ser Phe Gly Arg Pro Met Trp Asp Asn Glu435 440 445 Phe Ser Arg Ile Ala Thr Ala His Pro Lys Ser Val Val Gly ValPhe 450 455 460 Leu Cys Gly Pro Pro Thr Leu Ala Lys Ser Leu Arg Lys CysCys Arg 465 470 475 480 Arg Tyr Ser Ser Leu Asp Pro Arg Lys Val Gln PheTyr Phe Asn Lys 485 490 495 Glu Thr Phe 43 35 DNA Artificial SequenceDescription of Artificial Sequence synthetic primer 43 ttctgagtaggtgtgcattt gagtgtcata aagac 35 44 45 DNA Artificial Sequence Descriptionof Artificial Sequence synthetic primer 44 ttttccgtca aaattataactttttatttt ctttttataa cacat 45 45 5494 DNA Homo sapiens CDS(155)..(4810) 45 gcagagctgc agaggcaccg gacgagagag ggctccgcgg gcccagctggcagccaggcc 60 ggagacaagt tgcagtcccg ggctctggtg acgccgtggc cgcagggtctccattttggg 120 acattctaat ccctgagccc ctattatttt catc atg ggc ttc tgc ctggct cta 175 Met Gly Phe Cys Leu Ala Leu 1 5 gca tgg aca ctt ctg gtt ggggca tgg acc cct ctg gga gct cag aac 223 Ala Trp Thr Leu Leu Val Gly AlaTrp Thr Pro Leu Gly Ala Gln Asn 10 15 20 ccc att tcg tgg gag gtg cag cgattt gat ggg tgg tac aac aac ctc 271 Pro Ile Ser Trp Glu Val Gln Arg PheAsp Gly Trp Tyr Asn Asn Leu 25 30 35 atg gag cac aga tgg ggc agc aaa ggctcc cgg ctg cag cgc ctg gtc 319 Met Glu His Arg Trp Gly Ser Lys Gly SerArg Leu Gln Arg Leu Val 40 45 50 55 cca gcc agc tat gca gat ggc gtg taccag ccc ttg gga gaa ccc cac 367 Pro Ala Ser Tyr Ala Asp Gly Val Tyr GlnPro Leu Gly Glu Pro His 60 65 70 ctg ccc aac ccc cga gac ctt agc aac accatc tca agg ggc cct gca 415 Leu Pro Asn Pro Arg Asp Leu Ser Asn Thr IleSer Arg Gly Pro Ala 75 80 85 ggg ctg gcc tcc ctg aga aac cgc aca gtg ttgggg gtc ttc ttt ggc 463 Gly Leu Ala Ser Leu Arg Asn Arg Thr Val Leu GlyVal Phe Phe Gly 90 95 100 tat cac gtg ctt tca gac ctg gtg agc gtg gaaact ccc ggc tgc ccc 511 Tyr His Val Leu Ser Asp Leu Val Ser Val Glu ThrPro Gly Cys Pro 105 110 115 gcc gag ttc ctc aac att cgc atc ccg ccc ggagac ccc atg ttc gac 559 Ala Glu Phe Leu Asn Ile Arg Ile Pro Pro Gly AspPro Met Phe Asp 120 125 130 135 ccc gac cag cgc ggg gac gtg gtg ctg cccttc cag aga agc cgc tgg 607 Pro Asp Gln Arg Gly Asp Val Val Leu Pro PheGln Arg Ser Arg Trp 140 145 150 gac ccc gag acc gga cgg agt ccc agc aatccc cgg gac ccg gcc aac 655 Asp Pro Glu Thr Gly Arg Ser Pro Ser Asn ProArg Asp Pro Ala Asn 155 160 165 cag gtg acg ggc tgg ctg gac ggc agc gccatc tat ggt tcc tcg cat 703 Gln Val Thr Gly Trp Leu Asp Gly Ser Ala IleTyr Gly Ser Ser His 170 175 180 tcc tgg agc gac gcg ctg cgg agc ttc tccagg gga cag ctg gcg tcg 751 Ser Trp Ser Asp Ala Leu Arg Ser Phe Ser ArgGly Gln Leu Ala Ser 185 190 195 ggg ccc gac ccc gct ttt ccc cga gac tcgcag aac ccc ctg ctc atg 799 Gly Pro Asp Pro Ala Phe Pro Arg Asp Ser GlnAsn Pro Leu Leu Met 200 205 210 215 tgg gcg gcg ccc gac ccc gcc acc gggcag aac ggg ccc cgg ggg ctg 847 Trp Ala Ala Pro Asp Pro Ala Thr Gly GlnAsn Gly Pro Arg Gly Leu 220 225 230 tac gcc ttc ggg gca gag aga ggg aaccgg gaa ccc ttc ctg cag gcg 895 Tyr Ala Phe Gly Ala Glu Arg Gly Asn ArgGlu Pro Phe Leu Gln Ala 235 240 245 ctg ggc ctg ctc tgg ttc cgc tac cacaac ctg tgg gcg cag agg ctg 943 Leu Gly Leu Leu Trp Phe Arg Tyr His AsnLeu Trp Ala Gln Arg Leu 250 255 260 gcc cgc cag cac cca gac tgg gag gacgag gag ctg ttc cag cac gca 991 Ala Arg Gln His Pro Asp Trp Glu Asp GluGlu Leu Phe Gln His Ala 265 270 275 cgc aag agg gtc atc gcc acc tac cagaac atc gct gtg tat gag tgg 1039 Arg Lys Arg Val Ile Ala Thr Tyr Gln AsnIle Ala Val Tyr Glu Trp 280 285 290 295 ctg ccc agc ttc ctg cag aaa acactc ccg gag tat aca gga tac cgg 1087 Leu Pro Ser Phe Leu Gln Lys Thr LeuPro Glu Tyr Thr Gly Tyr Arg 300 305 310 cca ttt ctg gac ccc agc atc tcctca gag ttc gtg gcg gcc tct gag 1135 Pro Phe Leu Asp Pro Ser Ile Ser SerGlu Phe Val Ala Ala Ser Glu 315 320 325 cag ttc ctg tcc acc atg gtg ccccct ggc gtc tac atg aga aat gcc 1183 Gln Phe Leu Ser Thr Met Val Pro ProGly Val Tyr Met Arg Asn Ala 330 335 340 agc tgc cac ttc cag ggg gtc atcaat cgg aac tca agt gtc tcc aga 1231 Ser Cys His Phe Gln Gly Val Ile AsnArg Asn Ser Ser Val Ser Arg 345 350 355 gct ctc cgg gtc tgc aac agc tactgg agc cgt gag cac cca agc cta 1279 Ala Leu Arg Val Cys Asn Ser Tyr TrpSer Arg Glu His Pro Ser Leu 360 365 370 375 caa agt gct gaa gat gtg gatgca ctg ctg ctg ggc atg gcc tcc cag 1327 Gln Ser Ala Glu Asp Val Asp AlaLeu Leu Leu Gly Met Ala Ser Gln 380 385 390 atc gca gag cga gag gac catgtg ttg gtt gaa gat gtg cgg gat ttc 1375 Ile Ala Glu Arg Glu Asp His ValLeu Val Glu Asp Val Arg Asp Phe 395 400 405 tgg cct ggg cca ctg aag ttttcc cgc aca gac cac ctg gcc agc tgc 1423 Trp Pro Gly Pro Leu Lys Phe SerArg Thr Asp His Leu Ala Ser Cys 410 415 420 ctg cag cgg ggc cgg gat ctgggc ctg ccc tct tac acc aag gcc agg 1471 Leu Gln Arg Gly Arg Asp Leu GlyLeu Pro Ser Tyr Thr Lys Ala Arg 425 430 435 gca gca ctg ggc ttg tct cccatt acc cgc tgg cag gac atc aac cct 1519 Ala Ala Leu Gly Leu Ser Pro IleThr Arg Trp Gln Asp Ile Asn Pro 440 445 450 455 gca ctc tcc cgg agc aatgac act gta ctg gag gcc aca gct gcc ctg 1567 Ala Leu Ser Arg Ser Asn AspThr Val Leu Glu Ala Thr Ala Ala Leu 460 465 470 tac aac cag gac tta tcctgg cta gag ctg ctc cct ggg gga ctc ctg 1615 Tyr Asn Gln Asp Leu Ser TrpLeu Glu Leu Leu Pro Gly Gly Leu Leu 475 480 485 gag agc cac cgg gac cctgga cct ctg ttc agc acc atc gtc ctt gaa 1663 Glu Ser His Arg Asp Pro GlyPro Leu Phe Ser Thr Ile Val Leu Glu 490 495 500 caa ttt gtg cgg cta cgggat ggt gac cgc tac tgg ttt gag aac acc 1711 Gln Phe Val Arg Leu Arg AspGly Asp Arg Tyr Trp Phe Glu Asn Thr 505 510 515 agg aat ggg ctg ttc tccaag aag gag att gaa gaa atc cga aat acc 1759 Arg Asn Gly Leu Phe Ser LysLys Glu Ile Glu Glu Ile Arg Asn Thr 520 525 530 535 acc ctg cag gac gtgctg gtc gct gtt atc aac att gac ccc agt gct 1807 Thr Leu Gln Asp Val LeuVal Ala Val Ile Asn Ile Asp Pro Ser Ala 540 545 550 ctg cag ccc aat gtcttt gtc tgg cat aaa gga gac ccc tgt ccg cag 1855 Leu Gln Pro Asn Val PheVal Trp His Lys Gly Asp Pro Cys Pro Gln 555 560 565 ccg aga cag ctc agcact gaa ggc ctg cca gcg tgt gct ccc tct gtt 1903 Pro Arg Gln Leu Ser ThrGlu Gly Leu Pro Ala Cys Ala Pro Ser Val 570 575 580 gtt cgt gac tat tttgag ggc agt gga ttt ggc ttc ggg gtc acc atc 1951 Val Arg Asp Tyr Phe GluGly Ser Gly Phe Gly Phe Gly Val Thr Ile 585 590 595 ggg acc ctc tgt tgcttc cct ttg gtg agc ctg ctc agt gcc tgg att 1999 Gly Thr Leu Cys Cys PhePro Leu Val Ser Leu Leu Ser Ala Trp Ile 600 605 610 615 gtt gcc cgg ctccgg atg aga aat ttc aag agg ctc cag ggc cag gac 2047 Val Ala Arg Leu ArgMet Arg Asn Phe Lys Arg Leu Gln Gly Gln Asp 620 625 630 cgc cag agc atcgtg tct gag aag ctc gtg gga ggc atg gaa gct ttg 2095 Arg Gln Ser Ile ValSer Glu Lys Leu Val Gly Gly Met Glu Ala Leu 635 640 645 gaa tgg caa ggccac aag gag ccc tgc cgg ccc gtg ctt gtg tac ctg 2143 Glu Trp Gln Gly HisLys Glu Pro Cys Arg Pro Val Leu Val Tyr Leu 650 655 660 cag ccc ggg cagatc cgt gtg gta gat ggc agg ctc acc gtg ctc cgc 2191 Gln Pro Gly Gln IleArg Val Val Asp Gly Arg Leu Thr Val Leu Arg 665 670 675 acc atc cag ctgcag cct cca cag aag gtc aac ttc gtc ctg tcc agc 2239 Thr Ile Gln Leu GlnPro Pro Gln Lys Val Asn Phe Val Leu Ser Ser 680 685 690 695 aac cgt ggacgc cgc act ctg ctg ctc aag atc ccc aag gag tat gac 2287 Asn Arg Gly ArgArg Thr Leu Leu Leu Lys Ile Pro Lys Glu Tyr Asp 700 705 710 ctg gtg ctgctg ttt aac ttg gag gaa gag cgg cag gcg ctg gtg gaa 2335 Leu Val Leu LeuPhe Asn Leu Glu Glu Glu Arg Gln Ala Leu Val Glu 715 720 725 aat ctc cgggga gct ctg aag gag agc ggg ttg agc atc cag gag tgg 2383 Asn Leu Arg GlyAla Leu Lys Glu Ser Gly Leu Ser Ile Gln Glu Trp 730 735 740 gag ctg cgggag cag gag ctg atg aga gca gct gtg aca cgg gag cag 2431 Glu Leu Arg GluGln Glu Leu Met Arg Ala Ala Val Thr Arg Glu Gln 745 750 755 cgg agg cacctc ctg gag acc ttt ttc agg cac ctt ttc tcc cag gtg 2479 Arg Arg His LeuLeu Glu Thr Phe Phe Arg His Leu Phe Ser Gln Val 760 765 770 775 ctg gacatc aac cag gcc gac gca ggg acc ctg ccc ctg gac tcc tcc 2527 Leu Asp IleAsn Gln Ala Asp Ala Gly Thr Leu Pro Leu Asp Ser Ser 780 785 790 cag aaggtg cgg gag gcc ctg acc tgt gag ctg agc agg gcc gag ttt 2575 Gln Lys ValArg Glu Ala Leu Thr Cys Glu Leu Ser Arg Ala Glu Phe 795 800 805 gcc gagtcc ctg ggc ctc aag ccc cag gac atg ttt gtg gag tcc atg 2623 Ala Glu SerLeu Gly Leu Lys Pro Gln Asp Met Phe Val Glu Ser Met 810 815 820 ttc tctctg gct gac aag gat ggc aat ggc tac ctg tcc ttc cga gag 2671 Phe Ser LeuAla Asp Lys Asp Gly Asn Gly Tyr Leu Ser Phe Arg Glu 825 830 835 ttc ctggac atc ctg gtg gtc ttc atg aaa ggc tct cct gag gaa aag 2719 Phe Leu AspIle Leu Val Val Phe Met Lys Gly Ser Pro Glu Glu Lys 840 845 850 855 tctcgc ctt atg ttc cgc atg tac gac ttt gat ggg aat ggc ctc att 2767 Ser ArgLeu Met Phe Arg Met Tyr Asp Phe Asp Gly Asn Gly Leu Ile 860 865 870 tccaag gat gag ttc atc agg atg ctg aga tcc ttc atc gag atc tcc 2815 Ser LysAsp Glu Phe Ile Arg Met Leu Arg Ser Phe Ile Glu Ile Ser 875 880 885 aacaac tgc ctg tcc aag gcc cag ctg gct gag gtg gtg gag tcc atg 2863 Asn AsnCys Leu Ser Lys Ala Gln Leu Ala Glu Val Val Glu Ser Met 890 895 900 ttccgg gag tcg gga ttc cag gac aag gag gaa ctg aca tgg gaa gat 2911 Phe ArgGlu Ser Gly Phe Gln Asp Lys Glu Glu Leu Thr Trp Glu Asp 905 910 915 tttcac ttc atg ctg cgg gac cac aat agc gag ctc cgc ttc acg cag 2959 Phe HisPhe Met Leu Arg Asp His Asn Ser Glu Leu Arg Phe Thr Gln 920 925 930 935ctc tgt gtc aaa ggg gtg gag gtg cct gaa gtc atc aag gac ctc tgc 3007 LeuCys Val Lys Gly Val Glu Val Pro Glu Val Ile Lys Asp Leu Cys 940 945 950cgg cga gcc tcc tac atc agc cag gat atg atc tgt ccc tct ccc aga 3055 ArgArg Ala Ser Tyr Ile Ser Gln Asp Met Ile Cys Pro Ser Pro Arg 955 960 965gtg agt gcc cgc tgt tcc cgc agc gac att gag act gag ttg aca cct 3103 ValSer Ala Arg Cys Ser Arg Ser Asp Ile Glu Thr Glu Leu Thr Pro 970 975 980cag aga ctg cag tgc ccc atg gac aca gac cct ccc cag gag att cgg 3151 GlnArg Leu Gln Cys Pro Met Asp Thr Asp Pro Pro Gln Glu Ile Arg 985 990 995cgg agg ttt ggc aag aag gta acg tca ttc cag ccc ttg ctg ttc act 3199 ArgArg Phe Gly Lys Lys Val Thr Ser Phe Gln Pro Leu Leu Phe Thr 1000 10051010 1015 gag gcg cac cga gag aag ttc caa cgc agc tgt ctc cac cag acggtg 3247 Glu Ala His Arg Glu Lys Phe Gln Arg Ser Cys Leu His Gln Thr Val1020 1025 1030 caa cag ttc aag cgc ttc att gag aac tac cgg cgc cac atcggc tgc 3295 Gln Gln Phe Lys Arg Phe Ile Glu Asn Tyr Arg Arg His Ile GlyCys 1035 1040 1045 gtg gcc gtg ttc tac gcc atc gct ggg ggg ctt ttc ctggag agg gcc 3343 Val Ala Val Phe Tyr Ala Ile Ala Gly Gly Leu Phe Leu GluArg Ala 1050 1055 1060 tac tac tac gcc ttt gcc gca cat cac acg ggc atcacg gac acc acc 3391 Tyr Tyr Tyr Ala Phe Ala Ala His His Thr Gly Ile ThrAsp Thr Thr 1065 1070 1075 cgc gtg gga atc atc ctg tcg cgg ggc aca gcagcc agc atc tct ttc 3439 Arg Val Gly Ile Ile Leu Ser Arg Gly Thr Ala AlaSer Ile Ser Phe 1080 1085 1090 1095 atg ttc tcc tac atc ttg ctc acc atgtgc cgc aac ctc atc acc ttc 3487 Met Phe Ser Tyr Ile Leu Leu Thr Met CysArg Asn Leu Ile Thr Phe 1100 1105 1110 ctg cga gaa acc ttc ctc aac cgctac gtg ccc ttc gac gcc gcc gtg 3535 Leu Arg Glu Thr Phe Leu Asn Arg TyrVal Pro Phe Asp Ala Ala Val 1115 1120 1125 gac ttc cat cgc ctc att gcctcc acc gcc atc gtc ctc aca gtc tta 3583 Asp Phe His Arg Leu Ile Ala SerThr Ala Ile Val Leu Thr Val Leu 1130 1135 1140 cac agt gtg ggc cat gtggtg aat gtg tac ctg ttc tcc atc agc ccc 3631 His Ser Val Gly His Val ValAsn Val Tyr Leu Phe Ser Ile Ser Pro 1145 1150 1155 ctc agc gtc ctc tcttgc ctc ttt cct ggc ctc ttc cat gat gat ggg 3679 Leu Ser Val Leu Ser CysLeu Phe Pro Gly Leu Phe His Asp Asp Gly 1160 1165 1170 1175 tct gag ttcccc cag aag tat tac tgg tgg ttc ttc cag acc gta cca 3727 Ser Glu Phe ProGln Lys Tyr Tyr Trp Trp Phe Phe Gln Thr Val Pro 1180 1185 1190 ggc ctcacg ggg gtt gtg ctg ctc ctg atc ctg gcc atc atg tat gtc 3775 Gly Leu ThrGly Val Val Leu Leu Leu Ile Leu Ala Ile Met Tyr Val 1195 1200 1205 tttgcc tcc cac cac ttc cgc cgc cgc agt ttc cgg ggc ttc tgg ctg 3823 Phe AlaSer His His Phe Arg Arg Arg Ser Phe Arg Gly Phe Trp Leu 1210 1215 1220acc cac cac ctc tac atc ctg ctc tat gtc ctg ctc atc atc cat ggt 3871 ThrHis His Leu Tyr Ile Leu Leu Tyr Val Leu Leu Ile Ile His Gly 1225 12301235 agc ttt gcc ctg atc cag ctg ccc cgt ttc cac atc ttc ttc ctg gtc3919 Ser Phe Ala Leu Ile Gln Leu Pro Arg Phe His Ile Phe Phe Leu Val1240 1245 1250 1255 cca gca atc atc tat ggg ggc gac aag ctg gtg agc ctgagc cgg aag 3967 Pro Ala Ile Ile Tyr Gly Gly Asp Lys Leu Val Ser Leu SerArg Lys 1260 1265 1270 aag gtg gag atc agc gtg gtg aag gcg gag ctg ctgccc tca gga gtg 4015 Lys Val Glu Ile Ser Val Val Lys Ala Glu Leu Leu ProSer Gly Val 1275 1280 1285 acc cac ctg cgg ttc cag cgg ccc cag ggc tttgag tac aag tca ggg 4063 Thr His Leu Arg Phe Gln Arg Pro Gln Gly Phe GluTyr Lys Ser Gly 1290 1295 1300 cag tgg gtg cgg atc gct tgc ctg gct ctgggg acc acc gag tac cac 4111 Gln Trp Val Arg Ile Ala Cys Leu Ala Leu GlyThr Thr Glu Tyr His 1305 1310 1315 ccc ttc aca ctg acc tct gcg ccc catgag gac acg ctt agc ctg cac 4159 Pro Phe Thr Leu Thr Ser Ala Pro His GluAsp Thr Leu Ser Leu His 1320 1325 1330 1335 atc cgg gca gca ggg ccc tggacc act cgc ctc agg gag atc tac tca 4207 Ile Arg Ala Ala Gly Pro Trp ThrThr Arg Leu Arg Glu Ile Tyr Ser 1340 1345 1350 gcc ccg acg ggt gac agatgt gcc aga tac cca aag ctg tac ctt gat 4255 Ala Pro Thr Gly Asp Arg CysAla Arg Tyr Pro Lys Leu Tyr Leu Asp 1355 1360 1365 gga cca ttt gga gagggc cac cag gag tgg cat aag ttt gag gtg tca 4303 Gly Pro Phe Gly Glu GlyHis Gln Glu Trp His Lys Phe Glu Val Ser 1370 1375 1380 gtg tta gtg ggaggg ggc att ggg gtc acc cct ttt gcc tcc atc ctc 4351 Val Leu Val Gly GlyGly Ile Gly Val Thr Pro Phe Ala Ser Ile Leu 1385 1390 1395 aaa gac ctggtc ttc aag tca tcc gtc agc tgc caa gtg ttc tgt aag 4399 Lys Asp Leu ValPhe Lys Ser Ser Val Ser Cys Gln Val Phe Cys Lys 1400 1405 1410 1415 aagatc tac ttc atc tgg gtg acg cgg acc cag cgt cag ttt gag tgg 4447 Lys IleTyr Phe Ile Trp Val Thr Arg Thr Gln Arg Gln Phe Glu Trp 1420 1425 1430ctg gct gac atc atc cga gag gtg gag gag aat gac cac cag gac ctg 4495 LeuAla Asp Ile Ile Arg Glu Val Glu Glu Asn Asp His Gln Asp Leu 1435 14401445 gtg tct gtg cac atc tac atc acc cag ctg gct gag aag ttc gac ctc4543 Val Ser Val His Ile Tyr Ile Thr Gln Leu Ala Glu Lys Phe Asp Leu1450 1455 1460 agg acc act atg ctg tac atc tgt gag cgg cac ttc cag aaggtt ctg 4591 Arg Thr Thr Met Leu Tyr Ile Cys Glu Arg His Phe Gln Lys ValLeu 1465 1470 1475 aac cgg agt cta ttc aca ggc ctg cgc tcc atc acc cacttt ggc cgt 4639 Asn Arg Ser Leu Phe Thr Gly Leu Arg Ser Ile Thr His PheGly Arg 1480 1485 1490 1495 ccc ccc ttt gag ccc ttc ttc aac tcc ctg caggag gtc cac ccc cag 4687 Pro Pro Phe Glu Pro Phe Phe Asn Ser Leu Gln GluVal His Pro Gln 1500 1505 1510 gtc cgg aag atc ggg gtg ttt agc tgt ggcccc cct ggc atg acc aag 4735 Val Arg Lys Ile Gly Val Phe Ser Cys Gly ProPro Gly Met Thr Lys 1515 1520 1525 aat gtg gaa aag gcc tgt cag ctc atcaac agg cag gac cgg act cac 4783 Asn Val Glu Lys Ala Cys Gln Leu Ile AsnArg Gln Asp Arg Thr His 1530 1535 1540 ttc tcc cac cat tat gag aac ttctag gcccctgccc gggggttctg 4830 Phe Ser His His Tyr Glu Asn Phe 1545 1550cccactgtcc agttgagcag aggtttgagc ccacacctca cctctgttct tcctatttct 4890ggctgcctca gccttctctg atttcccacc tcccaacctt gttccaggtg gccatagtca 4950gtcaccatgt gtgggctcag ggacccccag gaccaggatg tgtctcagcc tggagaaatg 5010gtgggggggc agtgtctagg gactagagtg agaagtaggg gagctactga tttggggcaa 5070agtgaaacct ctgcttcaga cttcagaaac aaatctcaga agacaagctg acctgacaag 5130tactatgtgt gtgcatgtct gtatgtgtgt tggggcggtg agtgtaagga tgcagtggga 5190gcatggatgc tggcatctta gaaccctccc tactcccata cctcctcctc ttctgggctc 5250cccactgtca gacgggctgg caaatgcctt gcaggaggta gaggctggac ccatggcaag 5310ccatttacag aaacccactc ggcaccccag tctaacacca caactaattt cacccaaggt 5370tttaagcacg ttctttcatc agaccctggc ccaataccta tgtatgcaat gctcctcagc 5430cctcttctcc ctgctccagt agtctccctt ccaaataaat cacttttctg ccaaaaaaaa 5490aaaa 5494 46 1551 PRT Homo sapiens 46 Met Gly Phe Cys Leu Ala Leu AlaTrp Thr Leu Leu Val Gly Ala Trp 1 5 10 15 Thr Pro Leu Gly Ala Gln AsnPro Ile Ser Trp Glu Val Gln Arg Phe 20 25 30 Asp Gly Trp Tyr Asn Asn LeuMet Glu His Arg Trp Gly Ser Lys Gly 35 40 45 Ser Arg Leu Gln Arg Leu ValPro Ala Ser Tyr Ala Asp Gly Val Tyr 50 55 60 Gln Pro Leu Gly Glu Pro HisLeu Pro Asn Pro Arg Asp Leu Ser Asn 65 70 75 80 Thr Ile Ser Arg Gly ProAla Gly Leu Ala Ser Leu Arg Asn Arg Thr 85 90 95 Val Leu Gly Val Phe PheGly Tyr His Val Leu Ser Asp Leu Val Ser 100 105 110 Val Glu Thr Pro GlyCys Pro Ala Glu Phe Leu Asn Ile Arg Ile Pro 115 120 125 Pro Gly Asp ProMet Phe Asp Pro Asp Gln Arg Gly Asp Val Val Leu 130 135 140 Pro Phe GlnArg Ser Arg Trp Asp Pro Glu Thr Gly Arg Ser Pro Ser 145 150 155 160 AsnPro Arg Asp Pro Ala Asn Gln Val Thr Gly Trp Leu Asp Gly Ser 165 170 175Ala Ile Tyr Gly Ser Ser His Ser Trp Ser Asp Ala Leu Arg Ser Phe 180 185190 Ser Arg Gly Gln Leu Ala Ser Gly Pro Asp Pro Ala Phe Pro Arg Asp 195200 205 Ser Gln Asn Pro Leu Leu Met Trp Ala Ala Pro Asp Pro Ala Thr Gly210 215 220 Gln Asn Gly Pro Arg Gly Leu Tyr Ala Phe Gly Ala Glu Arg GlyAsn 225 230 235 240 Arg Glu Pro Phe Leu Gln Ala Leu Gly Leu Leu Trp PheArg Tyr His 245 250 255 Asn Leu Trp Ala Gln Arg Leu Ala Arg Gln His ProAsp Trp Glu Asp 260 265 270 Glu Glu Leu Phe Gln His Ala Arg Lys Arg ValIle Ala Thr Tyr Gln 275 280 285 Asn Ile Ala Val Tyr Glu Trp Leu Pro SerPhe Leu Gln Lys Thr Leu 290 295 300 Pro Glu Tyr Thr Gly Tyr Arg Pro PheLeu Asp Pro Ser Ile Ser Ser 305 310 315 320 Glu Phe Val Ala Ala Ser GluGln Phe Leu Ser Thr Met Val Pro Pro 325 330 335 Gly Val Tyr Met Arg AsnAla Ser Cys His Phe Gln Gly Val Ile Asn 340 345 350 Arg Asn Ser Ser ValSer Arg Ala Leu Arg Val Cys Asn Ser Tyr Trp 355 360 365 Ser Arg Glu HisPro Ser Leu Gln Ser Ala Glu Asp Val Asp Ala Leu 370 375 380 Leu Leu GlyMet Ala Ser Gln Ile Ala Glu Arg Glu Asp His Val Leu 385 390 395 400 ValGlu Asp Val Arg Asp Phe Trp Pro Gly Pro Leu Lys Phe Ser Arg 405 410 415Thr Asp His Leu Ala Ser Cys Leu Gln Arg Gly Arg Asp Leu Gly Leu 420 425430 Pro Ser Tyr Thr Lys Ala Arg Ala Ala Leu Gly Leu Ser Pro Ile Thr 435440 445 Arg Trp Gln Asp Ile Asn Pro Ala Leu Ser Arg Ser Asn Asp Thr Val450 455 460 Leu Glu Ala Thr Ala Ala Leu Tyr Asn Gln Asp Leu Ser Trp LeuGlu 465 470 475 480 Leu Leu Pro Gly Gly Leu Leu Glu Ser His Arg Asp ProGly Pro Leu 485 490 495 Phe Ser Thr Ile Val Leu Glu Gln Phe Val Arg LeuArg Asp Gly Asp 500 505 510 Arg Tyr Trp Phe Glu Asn Thr Arg Asn Gly LeuPhe Ser Lys Lys Glu 515 520 525 Ile Glu Glu Ile Arg Asn Thr Thr Leu GlnAsp Val Leu Val Ala Val 530 535 540 Ile Asn Ile Asp Pro Ser Ala Leu GlnPro Asn Val Phe Val Trp His 545 550 555 560 Lys Gly Asp Pro Cys Pro GlnPro Arg Gln Leu Ser Thr Glu Gly Leu 565 570 575 Pro Ala Cys Ala Pro SerVal Val Arg Asp Tyr Phe Glu Gly Ser Gly 580 585 590 Phe Gly Phe Gly ValThr Ile Gly Thr Leu Cys Cys Phe Pro Leu Val 595 600 605 Ser Leu Leu SerAla Trp Ile Val Ala Arg Leu Arg Met Arg Asn Phe 610 615 620 Lys Arg LeuGln Gly Gln Asp Arg Gln Ser Ile Val Ser Glu Lys Leu 625 630 635 640 ValGly Gly Met Glu Ala Leu Glu Trp Gln Gly His Lys Glu Pro Cys 645 650 655Arg Pro Val Leu Val Tyr Leu Gln Pro Gly Gln Ile Arg Val Val Asp 660 665670 Gly Arg Leu Thr Val Leu Arg Thr Ile Gln Leu Gln Pro Pro Gln Lys 675680 685 Val Asn Phe Val Leu Ser Ser Asn Arg Gly Arg Arg Thr Leu Leu Leu690 695 700 Lys Ile Pro Lys Glu Tyr Asp Leu Val Leu Leu Phe Asn Leu GluGlu 705 710 715 720 Glu Arg Gln Ala Leu Val Glu Asn Leu Arg Gly Ala LeuLys Glu Ser 725 730 735 Gly Leu Ser Ile Gln Glu Trp Glu Leu Arg Glu GlnGlu Leu Met Arg 740 745 750 Ala Ala Val Thr Arg Glu Gln Arg Arg His LeuLeu Glu Thr Phe Phe 755 760 765 Arg His Leu Phe Ser Gln Val Leu Asp IleAsn Gln Ala Asp Ala Gly 770 775 780 Thr Leu Pro Leu Asp Ser Ser Gln LysVal Arg Glu Ala Leu Thr Cys 785 790 795 800 Glu Leu Ser Arg Ala Glu PheAla Glu Ser Leu Gly Leu Lys Pro Gln 805 810 815 Asp Met Phe Val Glu SerMet Phe Ser Leu Ala Asp Lys Asp Gly Asn 820 825 830 Gly Tyr Leu Ser PheArg Glu Phe Leu Asp Ile Leu Val Val Phe Met 835 840 845 Lys Gly Ser ProGlu Glu Lys Ser Arg Leu Met Phe Arg Met Tyr Asp 850 855 860 Phe Asp GlyAsn Gly Leu Ile Ser Lys Asp Glu Phe Ile Arg Met Leu 865 870 875 880 ArgSer Phe Ile Glu Ile Ser Asn Asn Cys Leu Ser Lys Ala Gln Leu 885 890 895Ala Glu Val Val Glu Ser Met Phe Arg Glu Ser Gly Phe Gln Asp Lys 900 905910 Glu Glu Leu Thr Trp Glu Asp Phe His Phe Met Leu Arg Asp His Asn 915920 925 Ser Glu Leu Arg Phe Thr Gln Leu Cys Val Lys Gly Val Glu Val Pro930 935 940 Glu Val Ile Lys Asp Leu Cys Arg Arg Ala Ser Tyr Ile Ser GlnAsp 945 950 955 960 Met Ile Cys Pro Ser Pro Arg Val Ser Ala Arg Cys SerArg Ser Asp 965 970 975 Ile Glu Thr Glu Leu Thr Pro Gln Arg Leu Gln CysPro Met Asp Thr 980 985 990 Asp Pro Pro Gln Glu Ile Arg Arg Arg Phe GlyLys Lys Val Thr Ser 995 1000 1005 Phe Gln Pro Leu Leu Phe Thr Glu AlaHis Arg Glu Lys Phe Gln Arg 1010 1015 1020 Ser Cys Leu His Gln Thr ValGln Gln Phe Lys Arg Phe Ile Glu Asn 1025 1030 1035 1040 Tyr Arg Arg HisIle Gly Cys Val Ala Val Phe Tyr Ala Ile Ala Gly 1045 1050 1055 Gly LeuPhe Leu Glu Arg Ala Tyr Tyr Tyr Ala Phe Ala Ala His His 1060 1065 1070Thr Gly Ile Thr Asp Thr Thr Arg Val Gly Ile Ile Leu Ser Arg Gly 10751080 1085 Thr Ala Ala Ser Ile Ser Phe Met Phe Ser Tyr Ile Leu Leu ThrMet 1090 1095 1100 Cys Arg Asn Leu Ile Thr Phe Leu Arg Glu Thr Phe LeuAsn Arg Tyr 1105 1110 1115 1120 Val Pro Phe Asp Ala Ala Val Asp Phe HisArg Leu Ile Ala Ser Thr 1125 1130 1135 Ala Ile Val Leu Thr Val Leu HisSer Val Gly His Val Val Asn Val 1140 1145 1150 Tyr Leu Phe Ser Ile SerPro Leu Ser Val Leu Ser Cys Leu Phe Pro 1155 1160 1165 Gly Leu Phe HisAsp Asp Gly Ser Glu Phe Pro Gln Lys Tyr Tyr Trp 1170 1175 1180 Trp PhePhe Gln Thr Val Pro Gly Leu Thr Gly Val Val Leu Leu Leu 1185 1190 11951200 Ile Leu Ala Ile Met Tyr Val Phe Ala Ser His His Phe Arg Arg Arg1205 1210 1215 Ser Phe Arg Gly Phe Trp Leu Thr His His Leu Tyr Ile LeuLeu Tyr 1220 1225 1230 Val Leu Leu Ile Ile His Gly Ser Phe Ala Leu IleGln Leu Pro Arg 1235 1240 1245 Phe His Ile Phe Phe Leu Val Pro Ala IleIle Tyr Gly Gly Asp Lys 1250 1255 1260 Leu Val Ser Leu Ser Arg Lys LysVal Glu Ile Ser Val Val Lys Ala 1265 1270 1275 1280 Glu Leu Leu Pro SerGly Val Thr His Leu Arg Phe Gln Arg Pro Gln 1285 1290 1295 Gly Phe GluTyr Lys Ser Gly Gln Trp Val Arg Ile Ala Cys Leu Ala 1300 1305 1310 LeuGly Thr Thr Glu Tyr His Pro Phe Thr Leu Thr Ser Ala Pro His 1315 13201325 Glu Asp Thr Leu Ser Leu His Ile Arg Ala Ala Gly Pro Trp Thr Thr1330 1335 1340 Arg Leu Arg Glu Ile Tyr Ser Ala Pro Thr Gly Asp Arg CysAla Arg 1345 1350 1355 1360 Tyr Pro Lys Leu Tyr Leu Asp Gly Pro Phe GlyGlu Gly His Gln Glu 1365 1370 1375 Trp His Lys Phe Glu Val Ser Val LeuVal Gly Gly Gly Ile Gly Val 1380 1385 1390 Thr Pro Phe Ala Ser Ile LeuLys Asp Leu Val Phe Lys Ser Ser Val 1395 1400 1405 Ser Cys Gln Val PheCys Lys Lys Ile Tyr Phe Ile Trp Val Thr Arg 1410 1415 1420 Thr Gln ArgGln Phe Glu Trp Leu Ala Asp Ile Ile Arg Glu Val Glu 1425 1430 1435 1440Glu Asn Asp His Gln Asp Leu Val Ser Val His Ile Tyr Ile Thr Gln 14451450 1455 Leu Ala Glu Lys Phe Asp Leu Arg Thr Thr Met Leu Tyr Ile CysGlu 1460 1465 1470 Arg His Phe Gln Lys Val Leu Asn Arg Ser Leu Phe ThrGly Leu Arg 1475 1480 1485 Ser Ile Thr His Phe Gly Arg Pro Pro Phe GluPro Phe Phe Asn Ser 1490 1495 1500 Leu Gln Glu Val His Pro Gln Val ArgLys Ile Gly Val Phe Ser Cys 1505 1510 1515 1520 Gly Pro Pro Gly Met ThrLys Asn Val Glu Lys Ala Cys Gln Leu Ile 1525 1530 1535 Asn Arg Gln AspArg Thr His Phe Ser His His Tyr Glu Asn Phe 1540 1545 1550 47 3453 DNAHomo sapiens CDS (438)..(3134) 47 gtcctcgacc agtttgtacg gctgcgggatggtgaccgct actggtttga gaacaccagg 60 aatgggctgt tctccaagaa ggagattgagacatccgaaa taccaccgtg cgggacgtgc 120 tggtcgctgt tatcaacatt gaccccagtgccctgcagcc caatgtcttt gtctggcata 180 aaggtgcacc ctgccctcaa cctaagcagctcacaactga cggcctgccc cagtgtgcac 240 ccctgactgt gcttgacttc tttgaaggcagcagccctgg ttttgccatc accatcattg 300 ctctctgctg ccttccctta gtgagtctgcttctctctgg agtggtggcc tatttccggg 360 gccgagaaca caagaagcta caaaagaaactcaaagagag cgtgaagaag gaagcagcca 420 aagatggagt gccagcg atg gag tgg ccaggc ccc aag gag agg agc agt 470 Met Glu Trp Pro Gly Pro Lys Glu Arg SerSer 1 5 10 ccc atc atc atc cag ctg ctg tca gac agg tgt ctg cag gtc ctgaac 518 Pro Ile Ile Ile Gln Leu Leu Ser Asp Arg Cys Leu Gln Val Leu Asn15 20 25 agg cat ctc act gtg ctc cgt gtg gtc cag ctg cag cct ctg cag cag566 Arg His Leu Thr Val Leu Arg Val Val Gln Leu Gln Pro Leu Gln Gln 3035 40 gtc aac ctc atc ctg tcc aac aac cga gga tgc cgc acc ctg ctg ctc614 Val Asn Leu Ile Leu Ser Asn Asn Arg Gly Cys Arg Thr Leu Leu Leu 4550 55 aag atc cct aag gag tat gac ctg gtg ctg ctg ttt agt tct gaa gag662 Lys Ile Pro Lys Glu Tyr Asp Leu Val Leu Leu Phe Ser Ser Glu Glu 6065 70 75 gaa cgg ggc gcc ttt gtg cag cag cta tgg gac ttc tgc gtg cgc tgg710 Glu Arg Gly Ala Phe Val Gln Gln Leu Trp Asp Phe Cys Val Arg Trp 8085 90 gct ctg ggc ctc cat gtg gct gag atg agc gag aag gag cta ttt agg758 Ala Leu Gly Leu His Val Ala Glu Met Ser Glu Lys Glu Leu Phe Arg 95100 105 aag gct gtg aca aag cag cag cgg gaa cgc atc ctg gag atc ttc ttc806 Lys Ala Val Thr Lys Gln Gln Arg Glu Arg Ile Leu Glu Ile Phe Phe 110115 120 aga cac ctt ttt gct cag gtg ctg gac atc aac cag gcc gac gca ggg854 Arg His Leu Phe Ala Gln Val Leu Asp Ile Asn Gln Ala Asp Ala Gly 125130 135 acc ctg ccc ctg gac tcc tcc cag aag gtg cgg gag gcc ctg acc tgc902 Thr Leu Pro Leu Asp Ser Ser Gln Lys Val Arg Glu Ala Leu Thr Cys 140145 150 155 gag ctg agc agg gcc gag ttt gcc gag tcc ctg ggc ctc aag ccccag 950 Glu Leu Ser Arg Ala Glu Phe Ala Glu Ser Leu Gly Leu Lys Pro Gln160 165 170 gac atg ttt gtg gag tcc atg ttc tct ctg gct gac aag gat ggcaat 998 Asp Met Phe Val Glu Ser Met Phe Ser Leu Ala Asp Lys Asp Gly Asn175 180 185 ggc tac ctg tcc ttc cga gag ttc ctg gac atc ctg gtg gtc ttcatg 1046 Gly Tyr Leu Ser Phe Arg Glu Phe Leu Asp Ile Leu Val Val Phe Met190 195 200 aaa ggc tcc cca gag gat aag tcc cgt cta atg ttt acc atg tatgac 1094 Lys Gly Ser Pro Glu Asp Lys Ser Arg Leu Met Phe Thr Met Tyr Asp205 210 215 ctg gat gag aat ggc ttc ctc tcc aag gac gaa ttc ttc acc atgatg 1142 Leu Asp Glu Asn Gly Phe Leu Ser Lys Asp Glu Phe Phe Thr Met Met220 225 230 235 cga tcc ttc atc gag atc tcc aac aac tgc ctg tcc aag gcccag ctg 1190 Arg Ser Phe Ile Glu Ile Ser Asn Asn Cys Leu Ser Lys Ala GlnLeu 240 245 250 gcc gag gtg gtg gag tct atg ttc cgg gag tcg gga ttc caggac aag 1238 Ala Glu Val Val Glu Ser Met Phe Arg Glu Ser Gly Phe Gln AspLys 255 260 265 gag gag ctg aca tgg gag gat ttt cac ttc atg ctg cgg gaccat gac 1286 Glu Glu Leu Thr Trp Glu Asp Phe His Phe Met Leu Arg Asp HisAsp 270 275 280 agc gag ctc cgc ttc acg cag ctc tgt gtc aaa ggt gga ggtgga ggt 1334 Ser Glu Leu Arg Phe Thr Gln Leu Cys Val Lys Gly Gly Gly GlyGly 285 290 295 gga aat ggt att aga gat atc ttt aaa caa aac atc agc tgtcga gtc 1382 Gly Asn Gly Ile Arg Asp Ile Phe Lys Gln Asn Ile Ser Cys ArgVal 300 305 310 315 tcg ttc atc act cgg aca cct ggg gag cgc tcc cac ccccag gga ctg 1430 Ser Phe Ile Thr Arg Thr Pro Gly Glu Arg Ser His Pro GlnGly Leu 320 325 330 ggg ccc cct gtc cca gaa gcc cca gag ctg gga ggc cctgga ctg aag 1478 Gly Pro Pro Val Pro Glu Ala Pro Glu Leu Gly Gly Pro GlyLeu Lys 335 340 345 aag agg ttt ggc aaa aag gca gca gtg ccc act ccc cggctg tac aca 1526 Lys Arg Phe Gly Lys Lys Ala Ala Val Pro Thr Pro Arg LeuTyr Thr 350 355 360 gag gcg ctg caa gag aag atg cag cga ggc ttc cta gcccaa aag ctg 1574 Glu Ala Leu Gln Glu Lys Met Gln Arg Gly Phe Leu Ala GlnLys Leu 365 370 375 cag cag tac aag cgc ttc gtg gag aac tac cgg agg cacatc gtg tgt 1622 Gln Gln Tyr Lys Arg Phe Val Glu Asn Tyr Arg Arg His IleVal Cys 380 385 390 395 gtg gca atc ttc tcg gcc atc tgt gtt ggc gtg tttgca gat cgt gct 1670 Val Ala Ile Phe Ser Ala Ile Cys Val Gly Val Phe AlaAsp Arg Ala 400 405 410 tac tac tat ggc ttt gcc ttg cca ccc tcg gac attgca cag acc acc 1718 Tyr Tyr Tyr Gly Phe Ala Leu Pro Pro Ser Asp Ile AlaGln Thr Thr 415 420 425 ctc gtg ggc atc atc ctg tca cga ggc acg gcg gccagc gtc tcc ttc 1766 Leu Val Gly Ile Ile Leu Ser Arg Gly Thr Ala Ala SerVal Ser Phe 430 435 440 atg ttc tct tat atc ttg ctc acc atg tgc cgc aacctc ata acc ttc 1814 Met Phe Ser Tyr Ile Leu Leu Thr Met Cys Arg Asn LeuIle Thr Phe 445 450 455 ctg cga gag act ttc ctc aac cgc tat gtg cct tttgat gcc gca gtg 1862 Leu Arg Glu Thr Phe Leu Asn Arg Tyr Val Pro Phe AspAla Ala Val 460 465 470 475 gac ttc cac cgc tgg atc gcc atg gct gct gttgtc ctg gcc att ttg 1910 Asp Phe His Arg Trp Ile Ala Met Ala Ala Val ValLeu Ala Ile Leu 480 485 490 cac agt gct ggc cac gca gtc aat gtc tac atcttc tca gtc agc cca 1958 His Ser Ala Gly His Ala Val Asn Val Tyr Ile PheSer Val Ser Pro 495 500 505 ctc agc ctg ctg gcc tgc ata ttc ccc aac gtcttt gtg aat gat ggg 2006 Leu Ser Leu Leu Ala Cys Ile Phe Pro Asn Val PheVal Asn Asp Gly 510 515 520 tcc aag ctt ccc cag aag ttc tat tgg tgg ttcttc cag acc gtc cca 2054 Ser Lys Leu Pro Gln Lys Phe Tyr Trp Trp Phe PheGln Thr Val Pro 525 530 535 ggt atg aca ggt gtg ctt ctg ctc ctg gtc ctggcc atc atg tat gtc 2102 Gly Met Thr Gly Val Leu Leu Leu Leu Val Leu AlaIle Met Tyr Val 540 545 550 555 ttc gcc tcc cac cac ttc cgc cgc cgc agcttc cgg ggc ttc tgg ctg 2150 Phe Ala Ser His His Phe Arg Arg Arg Ser PheArg Gly Phe Trp Leu 560 565 570 acc cac cac ctc tac atc ctg ctc tat gccctg ctc atc atc cat ggc 2198 Thr His His Leu Tyr Ile Leu Leu Tyr Ala LeuLeu Ile Ile His Gly 575 580 585 agc tat gct ctg atc cag ctg ccc act ttccac atc tac ttc ctg gtc 2246 Ser Tyr Ala Leu Ile Gln Leu Pro Thr Phe HisIle Tyr Phe Leu Val 590 595 600 ccg gca atc atc tat gga ggt gac aag ctggtg agc ctg agc cgg aag 2294 Pro Ala Ile Ile Tyr Gly Gly Asp Lys Leu ValSer Leu Ser Arg Lys 605 610 615 aag gtg gag atc agc gtg gtg aag gcg gagctg ctg ccc tca gga gtg 2342 Lys Val Glu Ile Ser Val Val Lys Ala Glu LeuLeu Pro Ser Gly Val 620 625 630 635 acc tac ctg caa ttc cag agg ccc caaggc ttt gag tac aag tca gga 2390 Thr Tyr Leu Gln Phe Gln Arg Pro Gln GlyPhe Glu Tyr Lys Ser Gly 640 645 650 cag tgg gtg cgg atc gcc tgc ctg gctctg ggg acc acc gag tac cac 2438 Gln Trp Val Arg Ile Ala Cys Leu Ala LeuGly Thr Thr Glu Tyr His 655 660 665 ccc ttc aca ctg acc tcc gcg ccc catgag gac aca ctc agc ctg cac 2486 Pro Phe Thr Leu Thr Ser Ala Pro His GluAsp Thr Leu Ser Leu His 670 675 680 atc cgg gca gtg ggg ccc tgg acc actcgc ctc agg gag atc tac tca 2534 Ile Arg Ala Val Gly Pro Trp Thr Thr ArgLeu Arg Glu Ile Tyr Ser 685 690 695 tcc cca aag ggc aat ggc tgt gct ggatac cca aag ctg tac ctt gat 2582 Ser Pro Lys Gly Asn Gly Cys Ala Gly TyrPro Lys Leu Tyr Leu Asp 700 705 710 715 gga ccg ttt gga gag ggc cat caggag tgg cat aaa ttt gag gtg tca 2630 Gly Pro Phe Gly Glu Gly His Gln GluTrp His Lys Phe Glu Val Ser 720 725 730 gtg ttg gtg gga ggg ggc att ggggtc acc ccc ttt gcc tcc atc ctc 2678 Val Leu Val Gly Gly Gly Ile Gly ValThr Pro Phe Ala Ser Ile Leu 735 740 745 aaa gac ctg gtc ttc aag tca tccttg ggc agc caa atg ctg tgt aag 2726 Lys Asp Leu Val Phe Lys Ser Ser LeuGly Ser Gln Met Leu Cys Lys 750 755 760 aag atc tac ttc atc tgg gtg acacgg acc cag cgt cag ttt gag tgg 2774 Lys Ile Tyr Phe Ile Trp Val Thr ArgThr Gln Arg Gln Phe Glu Trp 765 770 775 ctg gct gac atc atc caa gag gtggag gag aac gac cac cag gac ctg 2822 Leu Ala Asp Ile Ile Gln Glu Val GluGlu Asn Asp His Gln Asp Leu 780 785 790 795 gtg tct gtg cac att tat gtcacc cag ctg gct gag aag ttc gac ctc 2870 Val Ser Val His Ile Tyr Val ThrGln Leu Ala Glu Lys Phe Asp Leu 800 805 810 agg acc acc atg cta tac atctgc gag cgg cac ttc cag aaa gtg ctg 2918 Arg Thr Thr Met Leu Tyr Ile CysGlu Arg His Phe Gln Lys Val Leu 815 820 825 aac cgg agt ctg ttc acg ggcctg cgc tcc atc acc cac ttt ggc cgt 2966 Asn Arg Ser Leu Phe Thr Gly LeuArg Ser Ile Thr His Phe Gly Arg 830 835 840 ccc ccc ttc gag ccc ttc ttcaac tcc ctg cag gag gtc cac cca cag 3014 Pro Pro Phe Glu Pro Phe Phe AsnSer Leu Gln Glu Val His Pro Gln 845 850 855 gtg cgc aag atc ggg gtg ttcagc tgc ggc cct cca gga atg acc aag 3062 Val Arg Lys Ile Gly Val Phe SerCys Gly Pro Pro Gly Met Thr Lys 860 865 870 875 aat gta gag aag gcc tgtcag ctc gtc aac agg cag gac cga gcc cac 3110 Asn Val Glu Lys Ala Cys GlnLeu Val Asn Arg Gln Asp Arg Ala His 880 885 890 ttc atg cac cac tat gagaac ttc tgagcctgtc ctccctggct gctgcttcca 3164 Phe Met His His Tyr GluAsn Phe 895 gtatcctgcc ttctcttctg tgcacctaag ttgcccagcc ctgctggcaatctctccatc 3224 agaatccacc ttaggcctca gctggagggc tgcagagccc ctcccaatattgggagaata 3284 ttgacccaga caattataca aatgagaaaa ggcattaaaa tttacgtttctgatgatggc 3344 aaagctcatt tttctattag taactctgct gaagatccat ttattgcaattcatgctgaa 3404 tctaaattgt aaaatttaaa attaaatgca tgtcctcaaa aaaaaaaaa3453 48 899 PRT Homo sapiens 48 Met Glu Trp Pro Gly Pro Lys Glu Arg SerSer Pro Ile Ile Ile Gln 1 5 10 15 Leu Leu Ser Asp Arg Cys Leu Gln ValLeu Asn Arg His Leu Thr Val 20 25 30 Leu Arg Val Val Gln Leu Gln Pro LeuGln Gln Val Asn Leu Ile Leu 35 40 45 Ser Asn Asn Arg Gly Cys Arg Thr LeuLeu Leu Lys Ile Pro Lys Glu 50 55 60 Tyr Asp Leu Val Leu Leu Phe Ser SerGlu Glu Glu Arg Gly Ala Phe 65 70 75 80 Val Gln Gln Leu Trp Asp Phe CysVal Arg Trp Ala Leu Gly Leu His 85 90 95 Val Ala Glu Met Ser Glu Lys GluLeu Phe Arg Lys Ala Val Thr Lys 100 105 110 Gln Gln Arg Glu Arg Ile LeuGlu Ile Phe Phe Arg His Leu Phe Ala 115 120 125 Gln Val Leu Asp Ile AsnGln Ala Asp Ala Gly Thr Leu Pro Leu Asp 130 135 140 Ser Ser Gln Lys ValArg Glu Ala Leu Thr Cys Glu Leu Ser Arg Ala 145 150 155 160 Glu Phe AlaGlu Ser Leu Gly Leu Lys Pro Gln Asp Met Phe Val Glu 165 170 175 Ser MetPhe Ser Leu Ala Asp Lys Asp Gly Asn Gly Tyr Leu Ser Phe 180 185 190 ArgGlu Phe Leu Asp Ile Leu Val Val Phe Met Lys Gly Ser Pro Glu 195 200 205Asp Lys Ser Arg Leu Met Phe Thr Met Tyr Asp Leu Asp Glu Asn Gly 210 215220 Phe Leu Ser Lys Asp Glu Phe Phe Thr Met Met Arg Ser Phe Ile Glu 225230 235 240 Ile Ser Asn Asn Cys Leu Ser Lys Ala Gln Leu Ala Glu Val ValGlu 245 250 255 Ser Met Phe Arg Glu Ser Gly Phe Gln Asp Lys Glu Glu LeuThr Trp 260 265 270 Glu Asp Phe His Phe Met Leu Arg Asp His Asp Ser GluLeu Arg Phe 275 280 285 Thr Gln Leu Cys Val Lys Gly Gly Gly Gly Gly GlyAsn Gly Ile Arg 290 295 300 Asp Ile Phe Lys Gln Asn Ile Ser Cys Arg ValSer Phe Ile Thr Arg 305 310 315 320 Thr Pro Gly Glu Arg Ser His Pro GlnGly Leu Gly Pro Pro Val Pro 325 330 335 Glu Ala Pro Glu Leu Gly Gly ProGly Leu Lys Lys Arg Phe Gly Lys 340 345 350 Lys Ala Ala Val Pro Thr ProArg Leu Tyr Thr Glu Ala Leu Gln Glu 355 360 365 Lys Met Gln Arg Gly PheLeu Ala Gln Lys Leu Gln Gln Tyr Lys Arg 370 375 380 Phe Val Glu Asn TyrArg Arg His Ile Val Cys Val Ala Ile Phe Ser 385 390 395 400 Ala Ile CysVal Gly Val Phe Ala Asp Arg Ala Tyr Tyr Tyr Gly Phe 405 410 415 Ala LeuPro Pro Ser Asp Ile Ala Gln Thr Thr Leu Val Gly Ile Ile 420 425 430 LeuSer Arg Gly Thr Ala Ala Ser Val Ser Phe Met Phe Ser Tyr Ile 435 440 445Leu Leu Thr Met Cys Arg Asn Leu Ile Thr Phe Leu Arg Glu Thr Phe 450 455460 Leu Asn Arg Tyr Val Pro Phe Asp Ala Ala Val Asp Phe His Arg Trp 465470 475 480 Ile Ala Met Ala Ala Val Val Leu Ala Ile Leu His Ser Ala GlyHis 485 490 495 Ala Val Asn Val Tyr Ile Phe Ser Val Ser Pro Leu Ser LeuLeu Ala 500 505 510 Cys Ile Phe Pro Asn Val Phe Val Asn Asp Gly Ser LysLeu Pro Gln 515 520 525 Lys Phe Tyr Trp Trp Phe Phe Gln Thr Val Pro GlyMet Thr Gly Val 530 535 540 Leu Leu Leu Leu Val Leu Ala Ile Met Tyr ValPhe Ala Ser His His 545 550 555 560 Phe Arg Arg Arg Ser Phe Arg Gly PheTrp Leu Thr His His Leu Tyr 565 570 575 Ile Leu Leu Tyr Ala Leu Leu IleIle His Gly Ser Tyr Ala Leu Ile 580 585 590 Gln Leu Pro Thr Phe His IleTyr Phe Leu Val Pro Ala Ile Ile Tyr 595 600 605 Gly Gly Asp Lys Leu ValSer Leu Ser Arg Lys Lys Val Glu Ile Ser 610 615 620 Val Val Lys Ala GluLeu Leu Pro Ser Gly Val Thr Tyr Leu Gln Phe 625 630 635 640 Gln Arg ProGln Gly Phe Glu Tyr Lys Ser Gly Gln Trp Val Arg Ile 645 650 655 Ala CysLeu Ala Leu Gly Thr Thr Glu Tyr His Pro Phe Thr Leu Thr 660 665 670 SerAla Pro His Glu Asp Thr Leu Ser Leu His Ile Arg Ala Val Gly 675 680 685Pro Trp Thr Thr Arg Leu Arg Glu Ile Tyr Ser Ser Pro Lys Gly Asn 690 695700 Gly Cys Ala Gly Tyr Pro Lys Leu Tyr Leu Asp Gly Pro Phe Gly Glu 705710 715 720 Gly His Gln Glu Trp His Lys Phe Glu Val Ser Val Leu Val GlyGly 725 730 735 Gly Ile Gly Val Thr Pro Phe Ala Ser Ile Leu Lys Asp LeuVal Phe 740 745 750 Lys Ser Ser Leu Gly Ser Gln Met Leu Cys Lys Lys IleTyr Phe Ile 755 760 765 Trp Val Thr Arg Thr Gln Arg Gln Phe Glu Trp LeuAla Asp Ile Ile 770 775 780 Gln Glu Val Glu Glu Asn Asp His Gln Asp LeuVal Ser Val His Ile 785 790 795 800 Tyr Val Thr Gln Leu Ala Glu Lys PheAsp Leu Arg Thr Thr Met Leu 805 810 815 Tyr Ile Cys Glu Arg His Phe GlnLys Val Leu Asn Arg Ser Leu Phe 820 825 830 Thr Gly Leu Arg Ser Ile ThrHis Phe Gly Arg Pro Pro Phe Glu Pro 835 840 845 Phe Phe Asn Ser Leu GlnGlu Val His Pro Gln Val Arg Lys Ile Gly 850 855 860 Val Phe Ser Cys GlyPro Pro Gly Met Thr Lys Asn Val Glu Lys Ala 865 870 875 880 Cys Gln LeuVal Asn Arg Gln Asp Arg Ala His Phe Met His His Tyr 885 890 895 Glu AsnPhe 49 26 DNA Artificial Sequence Description of Artificial SequencePrimer 49 cctgacagat gtatttcact acccag 26 50 24 DNA Artificial SequenceDescription of Artificial Sequence Primer 50 ggatcggagt cactcccttc gctg24 51 26 DNA Artificial Sequence Description of Artificial SequencePrimer 51 ctagaagctc tccttgttgt aataga 26 52 24 DNA Artificial SequenceDescription of Artificial Sequence Primer 52 atgaacacct ctggggtcag ctga24 53 24 DNA Artificial Sequence Description of Artificial SequencePrimer 53 atgaacacct ctggggtcag ctga 24 54 25 DNA Artificial SequenceDescription of Artificial Sequence Primer 54 gtcctctgca gcattgttcc tctta25 55 26 DNA Artificial Sequence Description of Artificial SequencePrimer 55 cctgacagat gtatttcact acccag 26 56 24 DNA Artificial SequenceDescription of Artificial Sequence Primer 56 ggatcggagt cactcccttc gctg24 57 25 DNA Artificial Sequence Description of Artificial SequencePrimer 57 aatgacactg tactggaggc cacag 25 58 24 DNA Artificial SequenceDescription of Artificial Sequence Primer 58 ctgccatcta ccacacggat ctgc24 59 24 DNA Artificial Sequence Description of Artificial SequencePrimer 59 cttgccattc caaagcttcc atgc 24 60 24 DNA Artificial SequenceDescription of Artificial Sequence Primer 60 gtacaagtca ggacagtggg tgcg24 61 24 DNA Artificial Sequence Description of Artificial SequencePrimer 61 tggatgatgt cagccagcca ctca 24

What is claimed is:
 1. An isolated protein with enzymatic activity ofgenerating reactive oxygen intermediates or a fragment thereof retainingsaid activity, wherein said protein comprises the amino acid sequence ofSEQ ID NO:2 or an amino acid sequence comprising additions, deletions orconservative substitutions of less than 5% of SEQ ID NO:2.
 2. Theisolated protein of claim 1, wherein said protein comprises the aminoacid sequence of SEQ ID NO:2, or a fragment of SEQ ID NO:2 havingenzymatic activity of generating reactive oxygen intermediates.
 3. Theisolated protein of claim 1, wherein the conservative substitutioncomprises substitution of: a) alanine, serine, or threonine for eachother; b) aspartic acid or glutamic acid for each other; c) asparagineor glutamine for each other; d) arginine or lysine for each other; e)isoleucine, leucine, methionine, or valine for each other; and f)phenylalanine, tyrosine, or tryptophan for each other.
 4. An isolatedprotein with enzymatic activity of generating reactive oxygenintermediates or a fragment thereof retaining said activity, whereinsaid protein comprises an amino acid sequence comprising additions,deletions or conservative substitutions of less than 5% of SEQ ID NO:2,wherein the conservative substitution comprises substitution of: a)alanine, serine, or threonine for each other; b) aspartic acid orglutamic acid for each other; c) asparagine or glutamine for each other;d) arginine or lysine for each other; e) isoleucine, leucine, methionineor valine for each other; f) phenylalanine, tyrosine, or tryptophan foreach other.
 5. An isolated protein comprising SEQ ID NO:2.
 6. Anisolated protein consisting of SEQ ID NO:2.